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SingleCellAnalysis
Methods and Protocols
Edited by Miodrag Gužvić
Department of Urology, University Hospital Regensburg, Regensburg, Germany
Editor
Miodrag Guz ˇ vic ´ Department of Urology
University Hospital Regensburg Regensburg, Germany
ISSN 1064-3745
Methods in Molecular Biology
ISSN 1940-6029 (electronic)
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Preface
An exciting time lies ahead for cellular biology. We are finally able to move away from assessing genome, transcriptome, proteome, or metabolome as a mean of a population and instead focus on the features of single cells. Developments in physics, chemistry, and molecular biology have pushed the boundaries of what is possible in cellular analysis to give unprecedented improvements in sensitivity and scale that are allowing us the capability to approach a full functional appraisal of single cells at a rapid rate.
The speed, sensitivity, accuracy, and scope of existing techniques for single-cell detection, isolation, and analysis (e.g., FACS, fluorescence microscopy, dielectrophoresis, whole genome and transcriptome amplification, high-throughput sequencing, etc.) have improved markedly, broadening research horizons, while complementary techniques in organic spectroscopy (e.g., MALDI imaging), inorganic spectroscopy (e.g., ICP-MS), and synchrotron analysis support this with more detailed information on aspects previously overlooked.
This volume summarizes these techniques, their capabilities, and the type of information that can be determined, and aims to give an overview of best practice for implementing them in single-cell analysis in an important and necessary move away from the bulk analysis that is constraining our boundaries.
Multi-omics analysis of single cells is gaining momentum in recent years. Such approaches are somewhat underrepresented in this book, and future editions should more comprehensively cover this important aspect of single-cell analysis. Furthermore, while an attempt was made to make this volume thematically comprehensive, it is somewhat biased toward the analysis of single cancer cells. The future editions of this book should broaden its scope by including protocols on isolation and analysis of single prokaryotic or plant cells. Still, many thematically overlapping or linked protocols presented in this volume enable development of complex and complete workflows to isolate and analyze single cells, even beyond cancer research.
Regensburg, GermanyMiodrag Guz
Acknowledgments
I am grateful to Rob Hutchinson for support in early phases of preparation of this book, and to contributors and the publisher for their limitless patience, which I tested many times while wrestling many obstacles trying to bring this book to see the light of the day. This book is dedicated to Christoph A. Klein, who introduced me to the multiverse of single-cell analysis.
1 Single Cell Isolation from Surgically Resected Tissue Via Mechanical Dissociation Using TissueGrinder
Prama Pallavi and Stefan Scheuermann
2 Circulating Tumor Cell Enrichment and Single-Cell Isolation Combining the CellSearch® and DEPArray™ Systems
Cacilia Kostler, Bernhard Polzer, and Barbara Alberter
3 Isolation of Viable Epithelial and Mesenchymal Circulating Tumor Cells from Breast Cancer Patients
Justyna Topa, Anna J. Z ˙ aczek, and Aleksandra Markiewicz
4 Single-Cell Recovery from Tumor Cell Xenotransplanted Zebrafish Embryos for the Study of Metastasis-Initiating Cells . .
Pablo Hurtado, Ine´s Martı ´ nez-Pena, and Roberto Pineiro
5 Isolation of Single Circulating Tumor Cells Using VyCAP Puncher System . .
Thais Pereira-Veiga, Bianca Behrens, Joska J. Broekmaat, Lisa Oomens, Michiel Stevens, Arjan G. J. Tibbe, Nikolas Stoecklein, Laura Muinelo-Romay, Roberto Pin ˜ eiro, and Clotilde Costa
6 Simultaneous Isolation and Amplification of mRNA and Genomic DNA of a Single Cell
Miodrag Guz ˇ vic ´
7 Isolation and Genomic Analysis of Circulating Tumor Cell Clusters in Cancer Patients 101 Carolina Reduzzi, Marta Vismara, Thomas Schamberger, Marco Silvestri, Rosita Motta, Bernhard M. Polzer, and Vera Cappelletti
8 Establishing Single-Cell Clones from In Vitro-Cultured Circulating Tumor Cells
Teng Teng and Min Yu
9 Immunofluorescence Combined with Single-Molecule RNA Fluorescence In Situ Hybridization for Concurrent Detection of Proteins and Transcripts in Stress Granules
Jakub Kochan and Mateusz Wawro
10 Highly Multiplexed and Simultaneous Characterization of Protein and RNA in Single Cells by Flow or Mass Cytometry Platforms Using Proximity Ligation Assay for RNA 143 Andrew D. Duckworth, Joseph R. Slupsky, and Nagesh Kalakonda
11 Array-Based Comparative Genomic Hybridization for the Detection of Copy Number Alterations in Single Cells
Giancarlo Feliciello, Zbigniew Tadeusz Czyz, and Bernhard M. Polzer
12
Single Cell Micro RNA Sequencing Library Preparation
Sarah M. Hu ¨ cker and Stefan Kirsch
13 Immunoblot Analysis from Single Cells Using Milo™ Single-Cell Western Platform
Prashant V. Thakkar
189
14 Imaging of Subcellular Distribution of Platinum in Single Cells Using Laser Ablation Inductively Coupled Plasma Mass Spectrometry 215
Amy J. Managh and Calum J. Greenhalgh
15 Patch-seq: Multimodal Profiling of Single-Cell Morphology, Electrophysiology, and Gene Expression
Cathryn R. Cadwell and Andreas S. Tolias
16 Single-Nucleus ATAC-seq for Mapping Chromatin Accessibility in Individual Cells of Murine Hearts
Michail Yekelchyk, Xiang Li, Stefan Guenther, and Thomas Braun
227
Contributors
BARBARA ALBERTER • Cellular and Molecular Diagnostics Group, Division of Personalized Cancer Therapy, Fraunhofer Institute of Toxicology and Experimental Medicine ITEM-R, Regensburg, Germany
BIANCA BEHRENS • Experimental Surgical Oncology, General, Visceral and Pediatric Surgery, University Hospital and Medical Faculty, Heinrich-Heine-University Du¨sseldorf, Du¨sseldorf, Germany
THOMAS BRAUN • Department of Cardiac Development and Remodelling, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany; German Centre for Cardiovascular Research (DZHK), Partner site Rhein-Main, Frankfurt am Main, Germany
JOSKA J. BROEKMAAT • VyCAP B.V, Deventer, The Netherlands
CATHRYN R. CADWELL • Departments of Neurological Surgery and Pathology, School of Medicine, University of California San Francisco, San Francisco, CA, USA
VERA CAPPELLETTI • Biomarkers Unit, Fondazione IRCCS, Istituto Nazionale dei Tumori di Milano, Milan, Italy
CLOTILDE COSTA • Roche-Chus Joint Unit, Translational Medical Oncology Group, Oncomet, Health Research Institute of Santiago de Compostela (IDIS), Santiago de Compostela, Spain; CIBERONC, Centro de Investigacion Biome´dica en Red Ca ´ ncer, Madrid, Spain
ZBIGNIEW TADEUSZ CZYZ • Experimental Medicine and Therapy Research, University Regensburg, Regensburg, Germany
ANDREW D. DUCKWORTH • Department of Molecular and Clinical Cancer Medicine, Institute of Systems, Molecular and Integrative Biology, University of Liverpool, Liverpool, UK
GIANCARLO FELICIELLO • Cellular and Molecular Diagnostics Group, Division of Personalized Cancer Therapy, Fraunhofer Institute of Toxicology and Experimental Medicine ITEM-R, Regensburg, Germany
CALUM J. GREENHALGH • Department of Chemistry, Loughborough University, Loughborough, UK
STEFAN GUENTHER • Department of Cardiac Development and Remodelling, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany
MIODRAG GUZ ˇ VIC ´ • Department of Urology, University Hospital Regensburg, Regensburg, Germany
SARAH M. HU ¨ CKER • Fraunhofer Institut fu¨r Toxikologie und Experimentelle Medizin, Abteilung Personalisierte Tumortherapie, Regensburg, Germany
PABLO HURTADO • Roche-Chus Joint Unit, Translational Medical Oncology Group, Gil Casares Hospital, Oncomet, Health Research Institute of Santiago de Compostela, Santiago de Compostela, Spain
NAGESH KALAKONDA • Department of Molecular and Clinical Cancer Medicine, Institute of Systems, Molecular and Integrative Biology, University of Liverpool, Liverpool, UK
STEFAN KIRSCH • Fraunhofer Institut fu¨r Toxikologie und Experimentelle Medizin, Abteilung Personalisierte Tumortherapie, Regensburg, Germany
JAKUB KOCHAN • Faculty of Biochemistry, Biophysics and Biotechnology, Department of Cell Biochemistry, Jagiellonian University, Krakow, Poland
CA CILIA KO ¨ STLER • Cellular and Molecular Diagnostics Group, Division of Personalized Cancer Therapy, Fraunhofer Institute of Toxicology and Experimental Medicine ITEM-R, Regensburg, Germany
XIANG LI • Department of Cardiac Development and Remodelling, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany
AMY J. MANAGH • Department of Chemistry, Loughborough University, Loughborough, UK
ALEKSANDRA MARKIEWICZ • Laboratory of Translational Oncology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Gdansk, Poland
INE ´ S MARTI ´ NEZ-PENA • Roche-Chus Joint Unit, Translational Medical Oncology Group, Gil Casares Hospital, Oncomet, Health Research Institute of Santiago de Compostela, Santiago de Compostela, Spain
ROSITA MOTTA • Biomarkers Unit, Fondazione IRCCS, Istituto Nazionale dei Tumori di Milano, Milan, Italy
LAURA MUINELO-ROMAY • Liquid Biopsy Analysis Unit, Translational Medical Oncology Group, Health Research Institute of Santiago de Santiago de Compostela (IDIS), Santiago de Compostela, Spain; CIBERONC, Centro de Investigacion Biome´dica en Red Ca ´ ncer, Madrid, Spain
LISA OOMENS • VyCAP B.V, Deventer, The Netherlands
PRAMA PALLAVI • Department of Surgery, Universitatsmedizin Mannheim, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
THAIS PEREIRA-VEIGA • Roche-Chus Joint Unit, Translational Medical Oncology Group, Oncomet, Health Research Institute of Santiago de Compostela (IDIS), Santiago de Compostela, Spain; Department of Tumor Biology, Center of Experimental Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany
ROBERTO PINEIRO • Roche-Chus Joint Unit, Translational Medical Oncology Group, Gil Casares Hospital, Oncomet, Health Research Institute of Santiago de Compostela, Santiago de Compostela, Spain; CIBERONC, Centro de Investigacion Biome´dica en Red Ca ´ ncer, Madrid, Spain
BERNHARD POLZER • Cellular and Molecular Diagnostics Group, Division of Personalized Cancer Therapy, Fraunhofer Institute of Toxicology and Experimental Medicine ITEM-R, Regensburg, Germany
BERNHARD M. POLZER • Cellular and Molecular Diagnostics Group, Division of Personalized Cancer Therapy, Fraunhofer Institute of Toxicology and Experimental Medicine ITEM-R, Regensburg, Germany
CAROLINA REDUZZI • Biomarkers Unit, Fondazione IRCCS, Istituto Nazionale dei Tumori di Milano, Milan, Italy; Division of Hematology/Oncology, Weill Cornell Medicine, New York, United States
THOMAS SCHAMBERGER • Experimental Medicine and Therapy Research, University Regensburg, Regensburg, Germany
STEFAN SCHEUERMANN • Clinical Health Technologies, Fraunhofer Institute for Manufacturing Engineering and Automation IPA, Mannheim, Germany
MARCO SILVESTRI • Biomarkers Unit, Fondazione IRCCS, Istituto Nazionale dei Tumori di Milano, Milan, Italy
JOSEPH R. SLUPSKY • Department of Molecular and Clinical Cancer Medicine, Institute of Systems, Molecular and Integrative Biology, University of Liverpool, Liverpool, UK
MICHIEL STEVENS • VyCAP B.V, Deventer, The Netherlands
NIKOLAS STOECKLEIN • Experimental Surgical Oncology, General, Visceral and Pediatric Surgery, University Hospital and Medical Faculty, Heinrich-Heine-University Du¨sseldorf, Du¨sseldorf, Germany
TENG TENG • Department of Stem Cell Biology and Regenerative Medicine, University of Southern California, Los Angeles, CA, USA; USC Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA
PRASHANT V. THAKKAR • Department of Hematology and Medical Oncology, Weill Cornell Medicine, New York, NY, USA; Department of Medicine, Weill Cornell Medicine, New York, NY, USA
ARJAN G. J. TIBBE • VyCAP B.V, Deventer, The Netherlands
ANDREAS S. TOLIAS • Department of Neuroscience, Baylor College of Medicine, Houston, TX, USA
JUSTYNA TOPA • Laboratory of Translational Oncology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Gdansk, Poland
MARTA VISMARA • Biomarkers Unit, Fondazione IRCCS, Istituto Nazionale dei Tumori di Milano, Milan, Italy
MATEUSZ WAWRO • Faculty of Biochemistry, Biophysics and Biotechnology, Department of Cell Biochemistry, Jagiellonian University, Krakow, Poland
MICHAIL YEKELCHYK • Department of Cardiac Development and Remodelling, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany
MIN YU • Department of Stem Cell Biology and Regenerative Medicine, University of Southern California, Los Angeles, CA, USA; USC Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA; Department of Pharmacology, University of Maryland School of Medicine, Baltimore, MD, USA
ANNA J. Z ˙ ACZEK • Laboratory of Translational Oncology, Intercollegiate Faculty of Biotechnology, University of Gdansk and Medical University of Gdansk, Gdansk, Poland
Chapter 1
Single Cell Isolation from Surgically Resected Tissue Via Mechanical Dissociation Using TissueGrinder
Prama Pallavi and Stefan Scheuermann
Abstract
Primary cells form the basis of modern-day in vitro research analysis tools. Many conventional procedures for generating single-cell suspensions from solid tissue are neither robust nor reproducible. Here we describe primary cells isolation from surgically resected tumor tissue via enzyme-free mechanical dissociation using TissueGrinder, a novel semi-automated benchtop device. The isolated cells can be used for any downstream biochemical or cell-based analytic assay.
Primary cells isolated from tissue are very important in vitro tools to answer several complex biological questions. In particular primary cells from surgically resected tissue can provide insight into cellular biological activity and hence new insights into disease [1]. Methods to isolate primary cells from tissues can be broadly divided into two main categories explant and tissue dissociation techniques using mechanical force and enzymatic digestion [2–5]. For more than 50 years, the explant method dominated the field of tissue culture for obtaining primary cells [6, 7]. In fact, this is one of the simplest techniques: the tissue is finely minced (1–2mm3) and placed in a culture flask [8], then the explant is covered with an appropriate cell outgrowth medium. Within a week’s time, cells migrate out of the explants and grow on the culture surface [9]. However, success is limited by various factors such as long processing times, low yield, and extensive manual workload, leading to a substantial increase in the overall time from tissue resection to the generation of a cell line. Furthermore, not all types of cells are able to migrate out of explant tissue. The second method, dissociation with enzymes, is not always desired because enzymatic digestion could affect the
proteins that might be later needed for labeling or staining, or are required for downstream molecular biological analysis [2]. In the mechanical approach, no additional enzymes or other agents are used to isolate cells from tissue, which could potentially alter the cells. During mechanical dissociation, however, the cells are exposed to a certain level of mechanical stress.
TissueGrinder technology enables the isolation of single cells from biological tissue of any origin by mechanical dissociation, minimizing the mechanical stress to preserve native character of cells to the maximum extent and aims for the utmost biological comparability with the sample donor. With this technology, a combination of shearing and cutting forces is applied to dissociate cells from pre-cut tissue [10]. The duration and speed of shearing and cutting forces can be optimized to develop tissue-and applicationspecific dissociation protocols. Manually diced tissue sample is transferred into grinding tubes, which contain an integrated grinding gear [10]. After dissociation, centrifugation of the grinding tubes allows easy collection of cells which can be either cultured directly or used for further analytical tests. By combining the process steps of dissociation with filtration and centrifugation in a one-step process, the risk of contamination can be minimized. Thus, TissueGrinder combines tissue-specific dissociation protocols with gentle mechanical dissociation to provide high-quality single-cell suspensions without the use of enzymes. Due to the high demand for primary cells and the limited availability of tissue samples, the isolation of primary cells should be as efficient as possible. In this method protocol, we describe a workflow for the generation of primary cell cultures using surgically resected tissue obtained from an anal cancer patient via TissueGrinder. The method can be applied also to other cancer tissue types [14].
2 Materials
2.1 Tissue Transport
Clinical tissue specimens are often stored under non-ideal conditions and for long time periods and can therefore be of poor quality at the time of processing. For this reason, it is important to ensure cooling of the specimen after resection and to process it promptly (see Notes).
1. Prepare centrifuge tube with 30 mL of DMEM medium supplemented with 100 units/mL of penicillin and 100 μg/mL of streptomycin.
2. Prepare a transport container with ice, to transport the tube with medium. Tissue is collected as quickly as possible with a cool box from surgery.
2.2 Dicing of the Tissue
2.3 Tissue Sample
Processing with the TissueGrinder
1. 10 cm diameter tissue culture grade petri dish.
2. Scalpels.
3. Cell culture hood.
4. Sterile PBS without Ca+2 and Mg+2
5. Sterile ammonium chloride solution: 0.8% NH4Cl, 0.1 mM EDTA in water, buffered with KHCO3 to achieve a final pH of 7.2–7.6.
6. Sterile pair of tweezers.
1. Weighing scale with precision of 1 mg.
2. 10 cm diameter tissue culture grade petri dish.
6. Benchtop centrifuge with swingout rotor available for 50 mL centrifuge tube.
7. Dresden medium: mixture of two thirds of the common DMEM (Sigma-Aldrich) supplemented with 10% FBS (Gibco) and 1% of penicillin (10,000 units/mL)/streptomycin (10,000 μg/mL) solution (Gibco) and one third of growth medium for Keratinocytes (Gibco) supplemented with Bovine Pituitary Extract (25 mg) and EGF Human Recombinant (2.5 μg) (Gibco). This medium was established through previous successes in obtaining cell lines from primary tissue [8].
8. Sterile serological pipettes (10 mL and 5 mL).
2.4 Cell Counting and Viability
2.5 Cell Seeding for Cell Culture
1. Luna counting slides.
2. Luna instrument.
3. Sterile Trypan Blue solution 0.4%.
Cell counting can be also done using Neubauer chamber and light microscope (see Notes).
1. Six-well plate tissue culture grade.
2. Culture medium.
Primary tumor cells isolated from primary tumors are cultured in “Dresden Medium.”
3. Sterile serological pipettes (10 mL and 5 mL).
3 Methods
3.1 Tissue Transport
3.2 Pre-Cutting of the Tissue Sample
1. Aliquot 20 mL of the DMEM supplemented with 1% of penicillin (10,000 units/mL)/streptomycin (10,000 μg/mL) solution in 50 mL centrifuge tube and place them in 4 °C fridge near operation theater. The amount of medium should be enough to submerge the tissue.
2. Place the surgically resected tumor tissue immediately in the centrifuge tube containing precooled transport medium (4 °C) and transport the tissue on ice from operation room to laboratory and process immediately (see Notes 1 and 2).
Note: All these steps should be performed under sterile tissue culture bench (see Notes 3 and 4).
1. Wash the tumor tissue with PBS supplemented with 1% Penicillin/Streptomycin solution (Penicillin (10,000 units/mL) and Streptomycin (10,000 μg/mL) solution)).
2. If the sample is still blood-soaked, then wash with an ammonium chloride solution to lyse erythrocytes.
3. Pre-cut the tissue into small cubes with an edge length of about 1–2 mm with help of a single-use scalpel.
4. Weigh up to 10–400 mg of the pre-cut tissue pieces in a clean and sterile petri dish.
3.3 Processing with the TissueGrinder
1. Take the disposable sterile grinding tube sets for the TissueGrinder. Assemble by latching the stator into the cell sieve. Next, wash stator and cell sieve with 1 mL of Dresden Medium and transfer the cell sieve to the sterile 50 mL centrifuge tube (see Note 5).
2. Place 10–400 mg of the prepared tissue pieces in the spare space between the grinding teeth of the rotor (using a 1 mL pipette) using a pair of sterile tweezers. Add 500 μl of the Dresden medium and place the stator with cell sieve on the rotor and close the tube. The detailed assembly of the grinding unit is shown in Fig. 1
3. Place the closed tube on a TissueGrinder benchtop port and select a predefined tissue dissociation program. There are three different generic programs soft, medium, and hard available for soft (easy to cut), medium (mildly fibroblastic), and hard tissues (highly fibroblastic and somewhat calcified). In addition, pre-installed programs for specific tissue types (such as liver, spleen, heart muscle, etc.) can be selected or customized for specific user applications. The generic programs should be selected and executed based on the texture of the
Fig. 1 Assembly of single-use TissueGrinder sterile grinding set (1) and the tissue dissociation workflow (2–12). The grinding set consists of a fitting lid to the 50 mL centrifuge tube with a centered hole, the rotor and the stator both forming the grinding gear, a standard cell strainer with a pore size of 100 μm, and a 50 mL centrifuge tube the assembled grinding set is locked with the rotor’s specific notch to its counterpart on the TissueGrinder. The diced tumor tissues, each with edge lengths of 1–2 mm and a total weight of 10–400 mg, are placed in the inner part of the rotor and the unit is assembled as shown under a cell culture bench. Diced tissue fragments are processed into single cells by alternating processes of grinding and cutting which is achieved in the TissueGrinder by clockwise or anticlockwise turning of the rotor. After the dissociation protocol, the tube is transferred directly to a standard laboratory centrifuge and the generated single-cell suspension is centrifuged through the 100 μm cell filter. The cell pellet is resuspended and transferred to a new vessel
tissue. A fine-tuning of the program to suit specific requirements can also be done. Table 1 describes predefined tissue dissociation programs.
4. Since anal cancer tissue is soft, run program soft. Following the program run, centrifuge the tube for 5 min at 350 g (see Notes/troubleshooting). After centrifugation, open the tube under laminar flow cabinet. Flush the tissue remains with 1 mL Dresden Medium through the cell sieve to ensure that all cells are collected in the 50 mL centrifugal tube. Discard the tissue fragments, which did not pass through the sieve.
5. Remove the cell sieve with the integrated stator from the tube. Close the lid of the tube and centrifuge again for 5 min at 350 g (see Note 6).
6. Resuspend cell pellet with 3 mL of Dresden Medium. Remove 50 μl of the resuspension in 1.5 mL microcentrifuge tube. This generated single-cell suspension can be used to supply various downstream applications (see Notes 7 and 8).
Table 2
Table 1
Steps of three TissueGrinder programs used for processing of tumor tissues
3.4 Cell Counting and Viability (see Notes 9–12)
A positive speed means clockwise turns which lead to a cutting of the tissue while a negative speed represents anticlockwise turns which causes a grinding of the tissue. This table is reproduced from [14]. The speed and duration for cutting and grinding can be set for each of the tissue processing programs
3.5 Cell Culture
1. Take 10 μl of the cell suspension and mix with 10 μl of the Trypan blue solution.
2. Load 10 μl of this cell solution on to Luna counting slide and inser t it in the Luna instrument to count the cells.
3. Note down the cell number (Table 2) and viability of the isolated cells.
1. Change the medium of the cells in the six well-plate twice a week until a confluent cell monolayer is observed.
2. Once a monolayer is observed (Fig. 2), sub-culture the cells into T25-and T75-flasks with passage ratio of 1:3 (see Notes 13 and 14).
Fig. 2 A representative image showing anal cancer cells isolated via TissueGrinder on the tenth day after processing. The images were taken with 4× Zeiss objective (LDA-Plan421231-991). The white scale bar represents 200 μm
4 Notes
1. A low temperature at around 4 °C helps to slow down metabolic activities that can lead to biochemical changes that alter gene expression, transcription, and the resulting protein profiles. This is especially important for the prevention of cell lysis.
2. Freeze/thaw cycles must also be avoided at this stage as cells can be very sensitive to temperature fluctuations, which can be very dramatic if the starting sample material is very limited.
3. Wear gloves when processing patient-derived tumor samples and avoid closing of scalpel to avoid injuries.
4. Surgically resected tissue from patients suffering from an active HIV-1, or other virologic infectious disease should be processed based on the biological safety level of the laboratory.
5. Sterile grinding kits for TissueGrinder are available with different sieve sizes. A sieve size of 70 μm should be used for cell isolation, while a size of 100 μm can be used to isolate clumps of cells which can be grown in Matrigel for microtissue culture.
6. Cells should always be washed in a buffer solution to remove cell debris, contaminants, and/or medium containing BSA/FCS. In addition, the centrifugation speed should be adapted to the cell type. The generally accepted recommendation for mammalian cells is to centrifuge at 300–900 g for 5 min to avoid cell disruption. Nonetheless, it’s crucial to be
aware that excessively low centrifugation speeds can result in the loss of cell pellets and, consequently, sample loss. Thus, great care should be exercised during the washing steps. Failed single cell analysis: Check cell viability and evaluate cell suspension for total cell yield, cell debris, and aggregates.
7. A multitude of applications and potential exploitation in the field of advanced purification of single-cell suspensions (Fluorescence-or magnetic activated cell sorting (FACS, MACS), laser capture microdissection (LCM), and microfluidic workflows) and single-cell analysis at genomic, transcriptomic, and proteomic levels are emerging [11].
8. Evaluation of a solid tissue-derived single-cell suspension is an important step prior to using cells for, e.g., flow cytometry. The three critical parameters to evaluate after mechanical dissociation of solid tissue are: (i) cell viability, (ii) absence of cell debris, and (iii) absence of aggregates [12]. The viability of cells in a single-cell suspension can be assessed using the trypan blue exclusion assay, in which dead cells incorporate trypan blue into their cytoplasm, while live cells retain their selectively permeable membrane and prevent trypan blue from entering the cytoplasm [13].
9. Cell viability in a single-cell suspension can easily be screened for using the trypan blue cell viability exclusion assay (Neubauer chamber).
10. Cell debris and cell aggregates can also quickly be evaluated using light microscopy.
11. In case of low cell yield, use polypropylene tubes and plates to avoid cell adhesion. Add washing steps.
12. In case of low cell viability, reduce the applied rotational speed of the TissueGrinder protocol by 50% and pipette gently while resuspending.
13. The isolated cells should be monitored every day for the first week to identify contaminated cultures.
14. Medium of the processed cultures should be changed every 3 days.
Acknowledgement
This research work was supported by Wilhelm Mu¨ller-Stiftung.
Ethical Statement This study was approved by the local ethics committee of the University Medical Center Mannheim (2012293 N-MA), and all of the donors gave their written informed consent.
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Circulating Tumor Cell Enrichment and Single-Cell Isolation Combining the CellSearch® and DEPArray™ Systems
C€ acilia Ko ¨ stler, Bernhard Polzer, and Barbara Alberter
Abstract
The analysis of circulating tumor cells (CTCs) has shown potential for detection of cancer spread, prognosis, therapeutic target selection, and monitoring of treatment response. CTCs can be obtained repeatedly by simple blood draws as so-called “liquid biopsy.” Thus, they can serve as a surrogate material for primary or metastatic tissue biopsies. In addition, isolation of CTCs provides the possibility to investigate those cells which may hold the (molecular) traits responsible for metastatic progression and ultimately patient death. As such, CTCs represent a target of utmost importance in cancer research and therapy. In this chapter, we describe a workflow for the enrichment of CTCs with the FDA-cleared CellSearch® system followed by the isolation of single CTCs using the DEPArray™ technology enabling further molecular single-cell analyses.
Key words CTC, CellSearch, DEPArray, Single-cell isolation, Dielectrophoresis, Liquid biopsy
1 Introduction
In most cancer patients, CTCs are low in abundance and heterogeneous in morphological and phenotypic profiles [1]. This complicates their enrichment and isolation for subsequent characterization [2]. However, a semi-automated and standardized workflow composed of CTC enrichment with the CellSearch® system and isolation of single CTCs with the DEPArray™ platform produces reliable results and can be applied as a routine workflow [3].
The CellSearch® system for the detection and enumeration of CTCs has been approved for diagnostic use in breast [4], prostate [5], and colorectal cancer [6]. The enrichment of CTCs with this platform makes use of the epithelial origin of carcinoma cells surrounded by mesenchymal cells of the blood compartment. Briefly, Epithelial Cell Adhesion Molecule (EpCAM)-expressing cells are enriched by antibody-coated ferromagnetic beads from a 7.5 mL blood sample and stained for intracellular cytokeratin proteins
8, 18, and 19 and for DAPI to determine the presence of nuclear DNA (positive identifiers), as well as for CD45 as a marker for contaminating blood leukocytes (negative identifier). Subsequently, the number of putative CTCs is determined automatically and verified by an experienced operator. Evaluation of CTC numbers at both early-and late-stage disease allows assessment of patient prognosis and is predictive of progression-free survival and overall survival [7, 8].
For research purposes, the precious patient sample analyzed by CellSearch® can be further utilized to isolate CTCs which can then be subjected to molecular analyses at the single-cell level. The DEPArray™ technology has proven beneficial as downstream single-cell isolation procedure. However, a mandatory buffer change before the CellSearch® sample can be transferred to the DEPArray™ cartridge is a critical step in terms of cell loss [2].
The DEPArray™ technology exploits the principles of dielectrophoresis (DEP) and the inducibility of electric dipoles in cells. After polarization of cells, they are trapped one by one in so-called electric “cages” on a chip. At this point, the cells can be detected by microscopic scanning of the whole sample with different channels simultaneously (brightfield and four fluorescence channels). Every event is recorded as an overlay image of the detected object (i.e., putative cell) and can be selected for subsequent separation. This is done by changing the electric field pattern of adjacent cages on the chip making selected cells move forward to recover them either as pools of cells or as single cells.
Once CTCs are isolated as single cells, their nucleic acids can be amplified and a multitude of molecular analyses can be performed, ranging from panel sequencing, copy number variation analysis to genome/exome sequencing.
2 Materials
2.1 CellSearch® System (see Fig. 1), Kits, and Additional Materials Required for Cell Enrichment
2.1.1 CELLTRACKS® AUTOPREP® System
2.1.2 CELLTRACKS ANALYZER II®
1. CELLTRACKS® AUTOPREP® System User Manual.
2. CELLTRACKS® AUTOPREP® System for enrichment, staining, and collection of the cells of interest.
3. MAGNEST® cartridge mounts.
4. Bottles with caps for waste, instrument buffer, de-ionized water, and cleaning solution.
5. Kit for the monthly maintenance.
6. Uninterrupted power supply.
1. CELLTRACKS ANALYZER II® User Manual.
2. CELLTRACKS ANALYZER II® for analysis of the enriched cells.
3. Two verification cartridges for the CELLTRACKS® System.
4. CELLTRACKS® justification cartridge.
5. Starter kit for lamp justification.
6. Mercury vapor lamp.
7. Uninterrupted power supply.
8. Local printer for reports.
9. Memorex® DVD + R to back-up results.
10. Fiber-free tissues.
2.1.3 CellSearch® Circulating Tumor Cell Kit
The CellSearch® Circulating Tumor Cell Kit (see Fig. 2) is used for capturing CTCs of epithelial origin (CD45-, EpCAM+, and cytokeratins 8+,18+, and/or 19+). It contains buffers and reagents for 16 enrichments. Up to its use it has to be stored at 2–8 °C(see Notes 1 and 2). Once it has been opened, the kit can be used for up to 30 days for diagnostic purposes (see Note 3). The kit contains the following reagents:
1. 3.0 mL Anti-EpCAM Ferrofluid (brown cap):
Suspension of 0.022% magnetic particles conjugated to a mouse monoclonal antibody specific for the cell surface marker EpCAM present on epithelial cells in a buffer with 0.03% bovine serum albumin (BSA) and 0.05% ProClin®
3. 3.0 mL Nucleic Acid Dye (blue cap): 0.005% 4′, 6-diamidino-2-phenylindole, dihydrochloride (DAPI), and 0.05% ProClin®300.
4. 3.0 mL Capture Enhancement Reagent (clear cap): 0.02% proprietary reagent for controlled ferrofluid aggregation, 0.5% BSA, and 0.1% sodium azide in buffer.
5. 3.0 mL Permeabilization Reagent (green cap): 0.011% proprietary permeabilization reagent and 0.1% sodium azide in buffer.
6. 3.0 mL Cell Fixative (red cap): 25% proprietary fixative ingredients, 0.1% BSA, and 0.1% sodium azide in buffer fixes cells for identification and enumeration.
7. 2 × 110 mL Dilution Buffer:
A buffer with 0.1% sodium azide is used for dilution. It is the only reagent of the kit, which should be stored at room temperature after opening the first time (see Note 2).
2.1.4 CellSearch® Circulating Tumor Cell Control Kit
The CellSearch® Circulating Tumor Cell Control Kit is used for testing system performance and operator technique. It is sufficient for 24 applications and proves that the system detects both high as well as low numbers of CTCs. It has to be stored at 2–8 °C and contains:
5. Protein gel loading tips round convenient for 200 μLmicropipettes.
6. 1.5 mL Protein LoBind tubes.
7. 50 mL screw cap tubes.
8. Racks for 1.5 mL and 50 mL tubes.
9. Micropipettes (10, 20, 200, 1000 μL) and related LoRetention filter tips (see Note 8).
10. Swinging bucket rotor centrifuge with adapters (see Note 9) for 1.5 mL tubes.
11. Laminar flow hood.
*Alternatively, the VR volume reduction device (Menarini Silicon Biosystems) can be used following the instructions of the user manual to reduce the volume prior to loading the DEPArray™ cartridge.
2.3 (Single)-Cell Isolation with the DEPArray™ Platform
1. DEPArray™ Plus for CTC Application Guide and DEPArray™ Instruction.
2. DEPArray™ system (see Fig. 3).
2.4 Volume Reduction of DEPArray™ Isolated Cells*
3. DEPArray™ cartridges.
4. Buffer for the DEPArray™ system (DABUF).
5. 1.5 mL Protein LoBind tubes.
6. MicroAmp Reaction Tubes with cap 0.2 mL.
7. Micropipettes 5000 μL, 20 μL and related LoRetention filter tips (see Note 8).
8. Ultrasonic bath with a pulse button.
9. Racks for 1.5 mL and 0.2 mL tubes.
10. Laminar flow hood.
11. Waterproof pen for the unique labeling of the tubes.
1. 0.2 mL MicroAmp Reaction tubes with the DEPArray™ isolated cells inside.
2. 1 × PBS.
3. 200 μL micropipette and related LoRetention filter tips (see Note 8).
4. Fixed rotor centrifuge with adapters for 0.2 mL tubes.
5. Racks for 0.2 mL tubes.
6. Freezer for storage.
*Alternatively, the VR volume reduction device (Menarini Silicon Biosystems) can be used following the instructions of the user manual to reduce the volume after the isolation of the cells.
3.1 CTC Enrichment with the CELLTRACKS® AUTOPREP® System
Before starting a run, the system must be cleaned (see Note 10). Eight samples can be processed in parallel. Make sure that the samples fulfill all requirements for the run (see Note 11). The necessity for a control sample arises whenever patient samples are processed or when using a new lot number of kit reagents (see Note 12). Make sure to wear the required protective clothes and gloves for all following steps.
1. Start the CellTracks® Autoprep® System.
2. Bring the CellSearch® Circulating Tumor Cell Kit and one bottle of the CellSearch® Circulating Tumor Cell Control Kit to room temperature (see Note 1).
3. Select “Run batch” and enter the password (see Note 13).
4. Make sure, that the system is clean. If necessary, perform the “Daily Cleaning Procedure” (see Note 10), otherwise go on with step 4
5. Take as many CellSearch®Conical Centrifuge Tubes and Caps as there are samples (including positive control if necessary). Put a barcode label (if available, see Note 14) on the CellSearch®Conical Centrifuge Tubes for the samples (see Note 15).
6. Mix the CellSave tube by inverting it five times (see Note 16).
7. Remove the cap of the CellSave tube and transfer 7.5 mL of the blood sample into a CellSearch® Conical Centrifuge Tube. Add 6.5 mL dilution buffer, close it with a cap and mix gently by inverting five times (see Note 16).
8. Repeat steps 6 and 7 for further samples.
9. Centrifuge the tubes with a horizontal swing-out style rotor at 800 g for 10 min at room temperature. Attention: Set break to “0” (see Note 17).
10. Prepare the positive control if necessary during the centrifugation step, otherwise switch to step 13.
11. Put an orange ID label onto the conical tube with the positive control (see Note 5).
12. Mix the 3 mL control tube gently by vortexing 5 s and inverting the bottle five times (see Note 18). Open the cap and transfer the complete volume also from the cap to a conical centrifuge tube (see Notes 19 and 20).
13. Open all bottles of the CellSearch® Circulating Tumor Cell Kit and inspect them (see Note 21).
14. Follow instructions on the screen for choosing the kit, selecting a marker and control.
15. After choosing the number of samples, including the control, the system is “homing,” which means being prepared for the run.
3.2 CTC Counting with the CELLTRACKS ANALYZER II® System
16. Place the rack with the CellSearch® Circulating Tumor Cell Kit into the CellTracks®AutoPrep® System (see Note 22) and confirm the kit data with “Next.”
17. Confirm, if results shall be used for patient management (see Note 23) or not. (If the kit is not valid anymore for in vitro diagnostics, see Note 3).
18. Put an empty CellSearch® cartridge (without lid) into the magnetic rack (MAGNEST®) and load it to the system (see Notes 24 and 25).
19. Repeat step 18 for all remaining MAGNEST® s.
20. Go on with loading the samples and/or control (see Note 26). If a control is included in the run, follow the loading order in analogy to the order of the MAGNEST®s which were loaded in step 18 (see Note 24).
21. Close the door and start the system with “Start.” During the r un, the estimated duration is indicated (see Note 27).
22. When the enrichment is finished, remove the MAGNEST® s from the instrument. Carefully inspect the content of the CellSearch® cartridge (see Note 28).
23. Store the MAGNEST®s with the filled and closed CellSearch® cartridge at room temperature at a dark place in a horizontal position and analyze them with the CELLTRACKS ANALYZER II® system within the next 24 h (see Note 29). This is described in Subheading 3.2.
24. Follow the instructions on the screen for removing the empty sample/control tubes and inspect them (see Note 30). Remove the kit and close the reagents (see Note 31).
25. Go on with the daily cleaning procedure if no further runs are planned.
1. Switch on the device, computer, and the mercury vapor lamp (see Note 32). The CELLTRACKS® software starts automatically.
2. Sign in with the unique password.
3. If it is necessary to perform a system verification (see Note 33), click the “QC test” folder symbol, clean the system verification cartridge with a fiber-free wipe, open the sample door, and load it to the sample rack. Push the “Start” button in the field “System verification” (see Note 34). After successful verification reload the verification cartridge.
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Indeed the passage is so monstrous, that one is almost inclined to think that it must have crept into the Talmud by mistake; or, at the least, to expect that it would be followed by reprehension the most explicit and severe. But no, a little lower down another of these “wise men” says,—
גדכ וערקל רתומ ץראה םע,
“It is lawful to rend an amhaaretz like a fish;” and, a little above, an Israelite is forbidden to marry the daughter of such a person, for that she is no better than a beast. But the whole of the preceding passage is so characteristic of the spirit of Rabbinism, that it is worth inserting—
׳וכו ןנבר ונת ,
“Our Rabbies have taught. Let a man sell all that he has, and marry the daughter of a learned man. If he cannot find the daughter of a learned man, let him take the daughter of the great men of the time. If he cannot find the daughter of a great man of the time, let him marry the daughter of the head of a congregation. If he cannot find the daughter of the head of a congregation, let him marry the daughter of an almoner. If he cannot find the daughter of an almoner, let him marry the daughter of a schoolmaster. But let him not marry the daughter of the unlearned, for they are an abomination, and their wives are vermin; and of their daughters it is said, ‘Cursed is he that lieth with any beast.’” Here, again, one is inclined to suppose that there is a mistake, or that these words were spoken in jest, though such a jest would be intolerably profane; but all ground for such supposition is removed on finding this passage transcribed into the digest of Jewish law, called the Schulchan Aruch, part 2; in the Hilchoth P’riah ur’viah, by which transcription it is stamped, with all the authority of a law. Here, then, the reader is led to think, that an amhaaretz must mean something more and worse than an unlearned man—that it ought, perhaps, to be taken in its literal signification, “people of the land,” and that it may refer to the idolatrous and wicked Canaanites. But the common usage of the Talmud forbids a supposition. There is a well-known sentence which shows that even a High Priest might be an amhaaretz:—
“A learned man, though illegitimate, goes before a High Priest, who is an amhaaretz.” Here the amhaaretz is plainly opposed to him that is learned. And so, on the page of the Talmud from which we have quoted above, we find the following words:—
“An amhaaretz is forbidden to eat the flesh of a beast, for it is said, ‘This is the law of the beast and the fowl.’ (Levit. xi. 46.) Every one that laboureth in the law, it is lawful for him to eat the flesh of the beast and the fowl. But for him who does not labour in the law, it is forbidden to eat the flesh of the beast and the fowl.” According to this passage an amhaaretz is one who does not labour in the study of the law; and it being found on the very same page with the above most revolting declarations, it plainly shows the proud and haughty spirit of the authors of the Talmud, and their utter contempt for the poor, whose circumstances preclude them from the advantages of study. But, in reading such passages, the question naturally suggests itself, to which of the two classes does the poor Jewish population of London belong? There must be at the least hundreds, if not thousands of poor Jews in this great city who cannot possibly devote themselves to study. Amongst whom, then, are they to be classed? Amongst the learned מימכח ידימלת? or amongst the unlearned ץראה ימע? Are they, their wives, and daughters, as the Talmud says, to be called an abomination, vermin, and compared to the beasts? Or can a religion inculcating such sentiments proceed from that Holy One who is no respecter of persons? See here, ye children of Abraham, whom the providence of God has placed amongst the children of poverty, and cut off from the advantage of a learned education. You are not disciples of the wise, nor the great men of the time, nor heads of synagogues, nor almoners, nor even schoolmasters. You are quite shut out from these classes whom your Talmudical doctors favour so highly. See, then, in the above passages, what the Talmud says of yourselves, your wives, and daughters? Can you believe that this is the law of the God of Israel?
Can you think for one moment, that these doctors knew “the old paths,” “the good way?” If you do we must assure you that we cannot. We rather find it in that book, which says, “Blessed is the man that considereth the poor and needy.” (Psalm xli. 1.) And in that other book, which speaks in the same spirit, and says that “God hath chosen the foolish things of this world to confound the wise; and the weak things of this world to confound the things which are mighty, and base things of the world, and things which are not, to bring to nought things that are; that no flesh should glory in his presence.” (1 Cor. i. 27, 28.)
No. II.
IMPLICIT FAITH NOT DUE TO THE RABBIES.
It appears from the undisguised acknowledgments of the New Testament, that the doctors and rabbies of the Jews, the Pharisees, and scribes, were the implacable enemies of Jesus of Nazareth, and that they were the main instruments in effecting his death. The modern Jews consider this fact as a sufficient apology for their rejection of his claims to the Messiahship. They take it for granted that the great and learned men of that day were also good men, and that they had valid reasons for their conduct. They think if Jesus of Nazareth had been the true Messiah, that the Sanhedrin, the great Jewish council of the time, would have acknowledged him, and conclude that, as they rejected him, he cannot be the true Messiah. The New Testament, on the contrary, accounts for their unbelief by plainly telling us, that they were bad men; and that they were enemies to the Lord Jesus, because he told them the truth, and exposed their hypocrisy. Now, which of these two representations accords with the truth? Were the scribes and Pharisees, those great advocates of the oral law, הפ לעבש הרות, good men or bad men? The readers of our first number will be in some degree qualified to answer this question. Could those be good men who profanely talked of the lawfulness of killing an unlearned man, and who contemptuously compared the wives and daughters of the unlearned to “vermin and beasts?” If they could talk with levity of “rending like a fish” an unlearned man, one of their own brethren who had never done them any harm, what were they likely to do with one who exposed their wickedness, and boldly told them that they by their traditions made void the law of God? The very fact, that Jesus of Nazareth was put to death by such men, is presumptive evidence,
that he was a good man, and that his claims were just. But, however that be, it is worth while to inquire into the charges, which the New Testament brings against these learned men, and to see whether they are substantiated by the memorials of their character and spirit, which they themselves have left us in their laws. One of the charges preferred against them is, that they were ambitious men, covetous of worldly honour, and loving the pre-eminence. “But all their works they do to be seen of men; they make broad their phylacteries, and enlarge the borders of their garments. And love the uppermost rooms at feasts, and the chief seats in the synagogues, and greetings in the markets, and to be called of men, Rabbi, Rabbi.” (Matt. xxiii. 5-7.) Now, is this charge true? Does the oral law justify this assertion, or does it prove, on the contrary, that the enemies of Jesus were humble, pious men, whose piety serves as a warrant for the uprightness of their conduct in their treatment of the Lord Jesus? Let the reader judge from the following laws which these men framed with respect to themselves. In the first place they claim for themselves more honour and reverence than is due to a man’s own parents:—
“As a man is commanded to honour and fear his father, so he is bound to honour and fear his Rabbi more than his father; for his father has been the means of bringing him into the life of this world, but his Rabbi, who teaches him wisdom, brings him to the life of the world to come.” (Hilchoth Talmud Torah, c. 5.) This general rule is bad enough, but the particulars are still worse. “If a man should see something that his father has lost, and something that his Rabbi has lost, he is first to return what his Rabbi has lost, and then to return that which belongs to his father. If his father and his Rabbi be oppressed with a load, he is first to help down that of his Rabbi, and then that of his father. If his father and his Rabbi be in captivity, he is first to ransom his Rabbi and afterwards his father unless his father be the disciple of a wise man (i.e., learned), in which case he may ransom his father first.” How fearful is this doctrine! A man is to see his father, the author of his existence, the guardian of his infancy,
who has laboured for his support, and watched over him in the hour of sickness, he is to see this friend, to whom, under God, he owes everything, pining away in the bitterness of captivity, and yet, when he has got the means of restoring him to liberty and his family, he is to leave him still in all his misery, and ransom the Rabbi; where is this written in the Old Testament? “Honour thy father and thy mother,” is there the first commandment that follows after our duty to God, and the first movement of natural affection. But this Rabbinical doctrine silences the voice of nature, and makes void the law of God. What is the doctrine of the New Testament here? “If any provide not for his own, and specially for those of his own house, he hath denied the faith, and is worse than an infidel.” (1 Tim. v. 8.) The disciples of the Lord Jesus Christ never claimed for themselves any honour like this. In the passage just cited, they plainly declare that the first, in the circle of duties to men, is the duty to our own flesh and blood. And the only case in which the New Testament permits a deviation from this rule, is that where the same exception is made in the law of Moses, when love to parents would interfere with love to God. “If any man come to me and hate not his father and mother, and wife and children, and brethren and sisters, yea, and his own life also, he cannot be my disciple.” (Luke xiv. 26.) Here father and mother, and kindred, are put in one category with a man’s own life, in order to show that there is but one case in which the natural ties of blood may be overlooked, and this is when the service of God requires it. As it is also written in the law of Moses, “If thy brother, the son of thy mother, or thy son, or thy daughter, or the wife of thy bosom, or thy friend who is as thine own soul, entice thee secretly, saying, Let us go and serve other gods, which thou hast not known, thou nor thy fathers.... Thou shalt not consent unto him, nor hearken unto him, neither shall thine eye pity him,” &c. (Deut. xiii. 6-9.) And thus the tribe of Levi is praised, because “He said unto his father and his mother, I have not known him; neither did he acknowledge his brethren, nor know his own children.” (Deut. xxxiii. 9.) But this Talmudical law is widely different. It has no saving clause to show that the case specified is an exception to the general rule. It does not pretend to suppose that the father is a bad man, or an idolater, or an apostate. It specifies but one exception, and that is, where the father
is “the disciple of a wise man;” otherwise, though he be a good man, and a pious man, a loving and tender parent, still he is to be disregarded by his own son, and the Rabbi preferred before him. Is it possible to doubt that the men who conceived, sanctioned, and promulgated a law like this, had an eye to their own personal honour and interest? Is it reasonable to suppose that men who would sacrifice their own father to the honour of their Rabbi, would be very tender about the life of one who appeared, like Jesus of Nazareth, as an opposer of their pretensions? Or can the Jews, with the law and the prophets in their hands, suppose that these men pointed to “the old paths,” “the good way?” This is certainly not the doctrine of Moses. He says:—
“Cursed be he that setteth light by his father or his mother, and all the people shall say, Amen.” (Deut. xxvii. 16.)
But these men did not stop here. They were not content with being exalted above father and mother. They did not scruple to assert, that their honour was as sacred as that of God himself:—
“Thou must consider no honour greater than the honour of the Rabbi, and no fear greater than the fear of the Rabbi. The wise men have said, The fear of thy Rabbi is as the fear of God.”
They endeavour to prove the validity of these extravagant claims by such passages as Exod. xvi. 8, “Your murmurings are not against us, but against the Lord.” But they have taken for granted what they can never prove, and that is, that every Rabbi is invested with the same office and authority as Moses. But where, in all the law of Moses, is there any warrant for such an assumption? Moses could with all propriety say, “Your murmurings are not against us, but against the Lord,” for he held a special commission from God, and had proved to the people the reality of his commission by a series of miracles. But this the Rabbies never pretended to do. In this dearth of evidence the advocates of tradition flee for refuge to Deut. xvii. 8, &c. “If there
arise a matter too hard for thee in judgment, between blood and blood, between plea and plea, and between stroke and stroke, being matters of controversy within thy gates; then shalt thou arise, and get thee up into the place which the Lord thy God shall choose; and thou shalt come unto the priests, the Levites, and unto the judge that shall be in those days, and inquire, and they shall show thee the sentence of judgment. And thou shalt do according to the sentence, which they of that place which the Lord shall choose shall shew thee; and thou shalt observe to do according to all that they inform thee; according to the sentence of the law which they shall teach thee, and according to the judgment which they shall tell thee, thou shalt do; thou shalt not decline from the sentence which they shall show thee to the right hand nor to the left.” Here, say the traditionists, is a plain and unequivocal command. No doubt, God here plainly declares what is to be done in a difficult case. He commands the Israelites to go to the place which the Lord God chose, that is, to the place where was found the ark of the covenant; and to inquire, not of the Rabbies, but of the priests, the Levites, and the judge טפושה. But this passage, instead of proving that “the fear of the Rabbi is as the fear of God,” proves the contrary. It supposes first, that the Rabbies and learned men may differ in judgment, that there may be a controversy, and consequently, that one party may be in the wrong. It, therefore, effectually overthrows Rabbinical infallibility. It shows that these learned men are, after all, only poor fallible creatures like ourselves, and that, therefore, we are not to fear them as we would fear God, nor reverence their dictates, as the Word of God. It shows secondly, that in a case of difficulty, the Israelites were not to appeal to the Rabbies, but to the priests םינהכ, and to the judge טפוש, and even to them only in the place which the Lord should choose. There is not one word said about the Rabbies or the wise men, and, therefore, this passage completely annihilates all their lofty pretensions. For centuries the place which the Lord chose has been desolate, and there has been no priest standing to minister before the Lord. The Jews have thus lost all possibility of appeal. They have neither ministering priest nor judge, and the Mosaic law nowhere recognises the pretensions of the Rabbies. But some Jew may say, that though this passage does not prove the authority of the Rabbies, it does at
least warrant the Jews in persisting to reject the claims of the Lord Jesus, for that he was condemned by the priests, and in Jerusalem, the place which the Lord chose. We confess that this objection is plausible; but can easily prove that it is nothing more. In order to this, we ask the Jews, whether the above command to abide by the sentence of the priests is in every case, and without any exception, binding? To this question there are two answers possible—Yes and No. If they say No, then they admit that the priests might sometimes be in the wrong, and we would, of course, take advantage of this admission to show that they erred in their judgment on Jesus of Nazareth. They will then, most probably, say, Yes; the sentence of the priests, the Levites, and the judges, is in every case binding, and Israel is commanded not to deviate from it, either to the right hand or to the left, upon pain of capital punishment. We beg of them then to turn to the 26th chapter of the Prophet Jeremiah, and to consider the case there set before them. We there find that Jeremiah had delivered a message from God, very similar to our Lord’s prediction of the destruction of Jerusalem. “I will make this house like Shiloh, and will make this city a curse to all the nations of the earth.” We find, further, that for this message the priests condemned Jeremiah to death, just as their successors condemned Jesus of Nazareth. “Now it came to pass, when Jeremiah had made an end of speaking all that the Lord had commanded him to speak unto all the people, that the priests, and the prophets, and all the people took him, saying, Thou shalt surely die.” We find, further, that this sentence was pronounced “in the place which the Lord had chosen,” in the Temple itself. “And all the people were gathered against Jeremiah in the house of the Lord.” We find, further, that the sentence against Jeremiah was no rash sudden act, but the deliberate judgment of the priests. For when the princes of Judah came afterwards to inquire into the matter, “Then spake the priests and the prophets unto the princes and to all the people, saying, This man is worthy to die, for he hath prophesied against this city, as ye have heard with your ears.” Now, then, we ask again, whether the people of Israel was in duty bound to abide by this sentence, and not to decline from it, either to the right hand or to the left? We fearlessly reply, that they were not bound by this sentence, and that, if they had executed it,
they would have been guilty of murder, as Jeremiah himself declares: “But know ye for certain, that if ye put me to death, ye shall surely bring innocent blood upon yourselves, and upon this city, and upon the inhabitants thereof: for of a truth the Lord hath sent me unto you to speak all these words in your ears.” We infer, therefore, that it was possible for the priests, assembled in solemn deliberation in the house of the Lord, to err in judgment, and to pronounce on unrighteous sentence. We infer, further, that it was possible for the priests so far to err, as to condemn to death a true prophet of the Lord. We infer, further, that in such a case the people was not bound by this mistaken judgment; but that it was their duty to decline from it, both to the right hand and to the left. We infer, lastly, that as the priests might mistake, and unjustly condemn to death a true prophet, their sentence against Jesus of Nazareth forms no more argument against the Messiahship of Jesus, than the similar sentence just considered did against the true prophetic character of Jeremiah; and that it affords just as little warrant for Jewish unbelief as the former sentence did for putting Jeremiah to death. But it may be asked, if the judgment of the priests was not infallible, and if men were sometimes justifiable in refusing it, what use was there in the above commandment to apply to them in cases of difficulty, and to abide by their sentence? The answer to this is very simple. The priest that stood to minister before the Lord had it in his power, before the destruction of the first Temple, to inquire of the Lord and to receive a miraculous answer from God himself, which answer was, of course, infallible, and universally obligatory, without the possibility of exception. We find in the Old Testament many instances in which the Israelites availed themselves of this power, as in Judges xx. 27, “And the children of Israel inquired of the Lord (for the ark of the covenant of God was there in those days: and Phinehas, the son of Eleazar, the son of Aaron, stood before it in those days), saving, Shall I yet again go out to battle against the children of Benjamin my brother, or shall I cease? And the Lord said, Go up; for to morrow I will deliver them into thine hand.” And in the history of David’s life, there are several instances of his employment of this miraculous power, as 1 Sam. xxiii. 4, “Then David inquired of the Lord yet again. And the Lord answered him and said, Arise, go
down to Keilah; for I will deliver the Philistines into thine hand.” In all such cases where the priest first inquired of the Lord, his sentence was, of course, infallible, and the Israelites were bound to abide by it. But where they did not inquire of the Lord, their sentence was only that of fallible men, and, therefore, not binding upon the consciences of the people. Of this sort was their sentence upon Jeremiah. Being wicked men, they did not choose to ask counsel of the Lord, but pronounced sentence according to the devices of their own hearts. In the case of the Lord Jesus Christ the priests could not ask counsel of the Lord, for in the second Temple the Urim and Thummim, and the ark of the covenant, were wanting; the miraculous power, therefore, did not exist, and for this very reason the sentence of the priests, during the whole period of the second Temple, was only fallible, like that of other men, and, therefore, not binding, and consequently of no force as an argument against the Messiahship of the Lord Jesus Christ. The above passage, therefore, from the 17th of Deuteronomy, is of no use to the Rabbinical Jews, it does not prove the infallibility of the priests in the second Temple, and is still less applicable for sanctioning the traditions of the oral law, and the extravagant claims of the Rabbies. Having given this passage the consideration it deserves, we now return to the laws which the Rabbies have made in favour of themselves, and for their own honour. We consider that the two passages of the oral law already quoted, prove that the New Testament gives a fair delineation of their character. When men, without any warrant from God’s Word, claim for themselves the same degree of reverence which is due to God, it must be admitted that they are vainglorious and wicked in no ordinary degree. But it is possible to descend to particulars:—For instance, our Lord says, that these men “loved greetings in the market-places, and to be called of men, Rabbi, Rabbi.” Now one of the laws, still extant, forbids a man, when speaking of his Rabbi, to call him by name:—
“It is forbidden to a disciple to call his Rabbi by name, even when he is not in his presence.” Another law, still extant, prescribes the formula of greeting or salutation:—
“Neither is he to salute his Rabbi, nor to return his salutation in the same manner that salutations are given or returned amongst friends. On the contrary, he is to bow down before the Rabbi, and to say to him, with reverence and honour, Peace be unto thee, Rabbi.” The Rabbinical Jews, who see this, must not mistake us. We do not consider it in anywise sinful, but decorous, to treat a Rabbi with all due respect. We should feel no objection ourselves to make a bow to a Rabbi, and to salute him in the prescribed formula. But we cite these laws to show that the New Testament gives a fair representation of the Pharisees: for men, who could gravely sit down and enter into all these details of the mode in which they were to be honoured, and then give out these laws as divine, and, besides all this, call in the civil power to enforce them, must have had no mean idea of themselves and their own dignity. It must never be forgotten that these laws are not the mere regulations of a religious community. When the Rabbies had the power in their own hands, they enforced them by civil sanctions. They were not satisfied with excluding despisers of Rabbinical authority from eternal life, they prosecuted such before the tribunals, and sentenced them to a pecuniary fine and excommunication, as may be seen from the following law:—
“Whosoever despises the wise men has no share in the world to come. But notwithstanding this, if there come witnesses to prove that he has been guilty of contempt, even in words, his sentence is excommunication, and the tribunal (house of judgment) excommunicates him publicly, and everywhere mulct him in a pound of gold, and give it to the wise man. He that despiseth a wise man in words, even after his death, is to be excommunicated by the tribunal,” &c. We now ask the Jews of modern times what they think
of those who made their own personal honour the subject of legislation, who required the same reverence for their words as the Word of God, and who dragged up him that refused it before a tribunal, had him sentenced to pecuniary fine, and excommunication; and, besides all this, excluded him from the hope of everlasting life? Had such men any idea of liberty of conscience?
No. III.
RABBINIC INJUSTICE TO WOMEN, SLAVES, AND GENTILES.
If any of our readers should think that the design of these papers is to represent the oral law as a system of unmixed evil, we beg to assure them that they are mistaken. We are fully aware that a system based on the law and the prophets, must and does contain much that is good and worthy of admiration. Of this nature is the general command to all Israelites to study the law, which is as follows:—“Every man of Israel is bound to study the law. Whether he be poor or rich, healthy or unhealthy, young or old, yea, though he live upon alms, and beg from door to door, and though he have a wife and children, he is bound to set apart a fixed time for the study of the law, by day and by night, as it is written, ‘Thou shalt meditate therein by day and by night,’” And again, the maxim, “Every one that is bound to learn is also bound to teach;” and that, “therefore, a man is bound to teach his son and his son’s son,” &c., is in accordance with the plain command of God, and is therefore good. But the explanation and development of these good principles shows that the system itself is radically bad, and therefore cannot be from God. No one will deny that the Rabbies are right in asserting the obligation resting on every Israelite to study the law: but they are wrong in their explanation of what the law is. Immediately after the above good command, the oral law goes on to say, “Every one is bound to divide the time of his study into three parts: one-third to be devoted to the written law; one-third to Mishna; and one-third to Gemara:” so that the written law of God is to have only half as much attention as the traditions of men. This is bad enough. But the Rabbies do not stop here. They go on to say, that this third of attention is only required
when a man begins to study, but that when he has made progress, he is to read the law of God only at times, and to devote himself to Gemara.
דומלל
“What has been said refers only to the beginning of a man’s learning, but as soon as a man becomes great in wisdom, and has no need of learning the written law, or of labouring constantly in the oral law, let him at fixed times read them, that he may not forget any of the judgments of the law, but let him devote all his days to Gemara.” It is to be observed that “oral law” is here taken in a limited sense, as referring to the expositions of the written law, or, as Rabbi Joseph Karo[2] explains it, the Mishna; and Gemara signifies the legal decisions which are inferred by a process of reasoning, and to this third topic of Jewish theology the Israelites are commanded to give the chief of their time and attention, rather than to the written Word of God.
The apparent excellence of the above command to study the law is thus utterly destroyed by the Rabbinical exposition of what is to be studied. And if we go on to inquire upon whom this command is binding, the Rabbinical answer will afford just as little satisfaction. When the Rabbies say, that “every man of Israel is bound to study the law,” they mean to limit the study to the men of Israel, and to exclude the women and slaves. The very first sentence of the Hilchoth Talmud Torah is
“Women and slaves and children are exempt from the study of the law.” According to this declaration, women are not obliged to learn. The following extract will confirm this opinion, and at the same time show that there is no obligation on fathers to have their daughters taught.
“A woman who learns the law has a reward, but it is not equal to the reward which the man has, because she is not commanded to do so: for no one who does anything which he is not commanded to do, receives the same reward as he who is commanded to do it, but a less one. But though the woman has a reward, the wise men have commanded that no man should teach his daughter the law, for this reason, that the majority of women have not got a mind fitted for study, but pervert the words of the law on account of the poverty of their mind. The wise men have said, Every one that teacheth his daughter the law is considered as if he taught her transgression.[3] But this applies only to the oral law. As to the written law, he is not to teach her systematically; but if he has taught her, he is not to be considered as having taught her transgression.”
According to this decision, it is absolutely forbidden to teach a woman the oral law; and the teaching of it is looked upon as the teaching of transgression תולפית. We cannot forbear asking the advocates of the oral law, whether it does not here testify against itself that it is bad. It declares of itself that it is unfit for the perusal and study of the pure female mind, and that it is as corrupting as the teaching of transgression. We ask, then, can such a law be divine? Can it proceed from the God of Israel, who hath said, “Be ye holy, for I am holy?” What a noble testimony to the superiority of the written Word, and to the justice of the Lord Jesus Christ’s opposition to the oral law! The oral law itself says, “He that teacheth his daughter the oral law, is to be considered as if he taught her transgression. He that teacheth her the written law, is not to be so considered.” With such a confession, we fearlessly ask the sons and daughters of Israel, who then was in the right? Jesus of Nazareth, who opposed it, or the scribes and Pharisees who defended it?
But “the wise men” also forbid Israelites to teach women the written law, and declare that women are not bound to learn. For the prohibition they assign two reasons. First, they say that God has commanded them to teach only their sons, in proof of which they refer to Deut. xi. 19, “And ye shall teach them your children.” In the Hebrew it is םכינב “your sons;” and the rabbies infer םכיתונב תא אלו, “and not your daughters.”[4] Secondly, they say, as we have seen above, “that the majority of women have not got minds fitted for study,” and in the Talmud[5] this is attempted to be proved from Scripture. “A wise woman once asked R. Eliezer, How it was that after the sin of the golden calf, those who were alike in transgressions did not all die the same death? He replied, A woman’s wisdom is only for the distaff, as it is written, ‘All the women that were wise-hearted did spin with their hands.’” (Exod. xxxv. 25.) We hesitate not to say, that both these reasons are contrary to Scripture. We do not deny that םכינב signifies sons, but we utterly deny the conclusion of the Rabbies, that because the masculine word is used, therefore the women are not included in the command. There is an abundance of instances in which the masculine word םינב is used for children generally, without any allusion to sex. Take for example Exod. xxii. 23 (in the English 24), “And my wrath shall wax hot, and I will kill you with the sword; and your wives shall be widows, and your children םכינב (literally your sons) orphans.” Here again the masculine word is used, so that if the Rabbinical argument be valid in the above case, it will be valid here, and consequently the daughters are excluded from this denunciation, so that the sons were to be orphans, but not the daughters, which is plainly impossible. In the same way we can prove that the daughters of Israel did not wander in the wilderness forty years, for in Numbers xiv. 33, it is said, “And your children םכינבו (literally your sons, and, therefore, according to Talmudic logic, not your daughters) shall wander in the wilderness forty years.” The same logic will also prove that during the three days of miraculous darkness in Egypt, the women of Israel were left in darkness as well as the Egyptians, for it is said all the children of Israel (לארשי ינב לכלו, literally the sons of Israel) had light in their dwellings. And thus also it might be proved that not one of the ten commandments is binding upon the women,
for the masculine gender is employed throughout. This logic, therefore, is evidently false; and we conclude, on the contrary, that as the women are included in all these passages—as they wandered through the wilderness, and had light in their dwellings—and are bound to keep the ten commandments as well as the men, so also they are included in the command, “Ye shall teach them your children,” and that, therefore, the command of the oral law not to teach women, is contrary to the Word of God. But we are not confined to argument, God has plainly commanded that the women should learn as well as the men. “And Moses commanded them, saying, At the end of every seven years, in the solemnity of the year of release in the Feast of Tabernacles, when all Israel is come to appear before the Lord thy God in the place which he shall choose, thou shalt read this law before all Israel in their hearing. Gather the people together, men and women, and children, and thy stranger that is within thy gates, that they may hear, and that they may learn ודמלי ןעמלו, and fear the Lord your God, and observe to do all the words of this law.” (Deut. xxxi. 10-12.) Here a most beautiful order is observed, and required of women as well as men; hearing—learning —fearing—keeping the words of the law—God wills that that women should fear him and keep his commandments as well as the men; and therefore he wills that they should make use of the same means, that they should hear, and learn all the words of the law. The traditionists have, therefore, in this case plainly made void the law of God. God commands women as well as men to learn the law; the Rabbies say they are exempt from this duty. God commands that the woman should be taught. It is plain, therefore, that the oral law, which contradicts the written law, cannot be from God. The command of God is so plain that it is unnecessary to enter deeply into the second Rabbinical reason for the prohibition to teach women the law. It is evident that God did not think that the poverty of their understanding was any obstacle to their learning his will. Indeed it has pleased Him to show that He is no respecter of persons with regard to male or female, more than with regard to rich or poor. He has not only given them his law, but conferred on women as well as men the gift of prophecy, so that the names of Deborah, Hannah, and Huldah, must ever be remembered amongst the inspired
messengers of God. The Rabbies seem to have forgotten that “the fear of the Lord is the beginning of wisdom,” and that this fear may be implanted by God just as easily in the heart of a woman as of a Rabbi. But without inquiring further into their reasons or their motives, suffice it to say, that the oral law in thus robbing women of their right and inheritance in the law of God, and in degrading them to the same category with children and slaves, is opposed to the plain commands of the written law. But not so the New Testament. It exactly agrees with the Old in considering woman as a rational and responsible being, and a candidate for everlasting life. It, therefore, gives one general rule for the education of children, male and female. “Ye fathers, provoke not your children to wrath, but bring them up in the nurture and admonition of the Lord.” (Ephes. vi. 4.) It does indeed prescribe modesty and subjection to the women in the mode or learning, but in so doing it plainly points out their duty to become acquainted with the will of God. “Let the woman learn in silence with all subjection. But I suffer not a woman to teach nor to usurp authority over the man, but to be in silence.” (1 Tim. ii. 11, 12.)
In these and other passages the woman is placed in the position assigned her in the Old Testament, and not in the very subordinate rank imposed upon her by the oral law. “Women, and slaves (םידבע), and children, are exempt from the study of the law.” But we think that this rule is as false with regard to slaves as to women. Here the oral law says that slaves are not bound to learn. In Hilchoth Avadim, c. viii. 18, we find that they are not to be taught.
“It is forbidden to a man to teach his slave the law.” But, alas, the passage of the Word of God which forbids it, is not referred to. It is only an inference from the passage, “Ye shall teach your sons;” but is evidently contrary to the whole tenour of the law of Moses. In the first place, the Israelite who had been sold by the tribunal, or who, on account of poverty, had sold himself, was still an Israelite, and did not forfeit, finally, his right to his inheritance in the land; how, then, could he forfeit his right to the law, which Moses gave as “the inheritance of the congregation of Jacob?” The law of Moses expressly provides a day of rest “for the man servant and the maid
servant,” that they may not only have rest for their bodies, but may have time to learn the will of God, and provide for that eternity to which they are hastening as well as their masters. Indeed, if meditation on the Word of God was more necessary for one Israelite than another, it was for the Hebrew servant. If he had been guilty of theft, and had been sold by the tribunal, he had special need of instruction in the law of God to lead him to repentance, and to teach him his duty for the future. If he had been guilty of no crime, but had been compelled by poverty to sacrifice his liberty, surely he needed the consolation which the Word of God can supply, to enable him to bear his hard lot with patience, and to prevent him from murmuring. But here the oral law steps in, and actually prohibits his master from teaching him; and instead of encouraging him in his leisure time to turn to the Word of God as his refuge and his comfort, it tells him that he is not bound to study it. Here, again, the New Testament is much more like the law of Moses, which breathes, all through, a spirit of the most tender compassion for those in servitude. Moses commands the Israelites to remember that they had themselves been bondmen in Egypt. The New Testament reminds Christian masters that they have a master in heaven. “Ye masters, do the same things unto them, forbearing threatening: knowing that your master also is in heaven; neither is there respect of persons with him.” (Ephes. vi. 9.) It also plainly teaches that the relation which exists between believing masters and servants is, before God, that of brethren. “And they that have believing masters, let them not despise them, because they are brethren; but rather do them service because they are faithful and beloved, partakers of the benefit.” (1 Tim. vi. 2.) Yea, the New Testament lays down a general principle, the very opposite of that, that “women, and slaves, and children are exempt from the study of the law.” It says, “There is neither Jew nor Greek, there is neither bond nor free, there is neither male nor female, for ye are all one in Christ Jesus.” (Gal. iii. 28.) It does not dispense men from their relative duties, nor deprive any of their legitimate privileges, but teaches that for all, Jew or Greek, bond or free, male or female, there is but one way of salvation. Very different is the doctrine of the oral law. We have seen that it makes a grand distinction between male and female, bond and free, we need not,
