We introduce a protocol for measuring real-time drug response in organotypic tumor tissue slices. The experimental strategy outlined here provides a platform to carry out medium-high throughput drug screens on tissue slices derived from clinical or mouse tumors in ex vivo conditions.
Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu constitutes the tumor microenvironment (TME) and can modulate response to therapy in vivo or drug response ex vivo. Conventional cancer drug discovery screens are carried out on cells cultured in a monolayer, a system critically lacking the influence of TME. Thus, experimental systems that integrate sensitive and high-throughput assays with physiological TME will strengthen the preclinical drug discovery process. Here, we introduce ex vivo tumor tissue slice culture as a platform for medium-high-throughput drug screening. Organotypic tissue slice culture constitutes precisely-cut, thin tumor sections that are maintained with the support of a porous membrane in a liquid-air interface. In this protocol, we describe the preparation and maintenance of tissue slices prepared from mouse tumors and tumors from patient-derived xenograft (PDX) models. To assess changes in tissue viability in response to drug treatment, we leveraged a biocompatible luminescence-based viability assay that enables real-time, rapid, and sensitive measurement of viable cells in the tissue. Using this platform, we evaluated dose-dependent responses of tissue slices to the multi-kinase inhibitor, staurosporine, and cytotoxic agent, doxorubicin. Further, we demonstrate the application of tissue slices for ex vivo pharmacology by screening 17 clinical and preclinical drugs on tissue slices prepared from a single PDX tumor. Our physiologically-relevant, highly-sensitive, and robust ex vivo screening platform will greatly strengthen preclinical oncology drug discovery and treatment decision making.
Cancer cell interactions with the physical and biochemical properties of the surrounding stromal tissue forms the TME. TME can stimulate tumor growth, metastasis, and modulate tumor response to therapy1. In conventional preclinical drug development, drug candidates are typically first screened using cultured cancer cell lines, an assay platform that critically lacks the TME2. This lack of physiological TME in cell-based prescreening stages may limit the discovery of effective agents in tumor-bearing animal models and may contribute to the high attrition rate of many promising oncology drugs in later clinical stages of development3.
Despite the importance of TME in modulating tumor drug responses, experimental constraints limit the application of more physiologically relevant systems during the early stages of drug discovery and development. It is impractical to screen hundreds of therapeutic agents on tumors from animal models or patient tumor specimens. Indeed, surgical specimens are scarce resources with varied genetic backgrounds and screening thousands of candidate molecules in animal models is not feasible due to experimental scale, cost, and animal welfare.
Tumor tissue slice culture, where precisely cut, thin tumor sections are cultured ex vivo, can address the limitation of physiological TME in drug screening assays. Historically, the field of neuroscience pioneered and made extensive optimizations of slice culture for brain tissue4. Recently, many studies have demonstrated preparation of slices from various types of tumor tissue including cell line-derived tumors, spontaneous tumor models, patient-derived xenografts (PDX), and primary patient tumors. Ex vivo tissue slice culture integrates benefits from both in vivo and in vitro culture5. Tumor tissue slices retain intact tissue architecture and variegated cell composition, enabling the study of cancer cells within the TME context.
This protocol introduces an organotypic tumor tissue slice culture system combined with a real-time, highly sensitive viability assay to evaluate drug responses. Drug efficacy tests on organotypic tumor tissue slices in previously introduced protocols rely on measuring changes in cell viability by fluorescent dye incorporation, immunohistochemistry (IHC), or MTT ((3- [4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide) assay6,7,8,9. However, all of these methods are end point assays and suffer from low sensitivity, long processing time, complex data analyses, narrow signal range, and high experimental error. Our luminescence-based live cell-compatible reagent improves these assays by providing a wide signal range and instantaneous (~5 min) measurement without prior processing and minimal post processing. This reagent is highly sensitive and can coexist in the cell culture media, allowing continuous and time-course measurement of cell viability. This assay system is applicable to high-throughput screening of drug candidates on tissue slices in preclinical drug development.
All mouse experiments were performed according to the recommendations in the Guide for the Care and Use of Laboratory Animals of the Animal Welfare Act and the National Institutes of Health guidelines for the care and use of animals in biomedical research.
1. Preparation of tumor tissue slices
- Prepare tissue slice culture (TSC) medium following the recipe in Table 1. Filter the medium with a 0.45 µm vacuum filter unit to sterilize.
- Aliquot 250 µL of TSC medium per well for a 24 well plate, or 1 mL of medium per well for a 6 well plate. Keep the plate in a 37 °C, 5% CO2 humidified incubator until use.
NOTE: The medium and supplements can be optimized depending on tissue types and experimental goals. The amount of medium should be just enough to soak the porous membrane of a culture insert. Do not exceed the level of the membrane. Adjust the medium volume if the medium level is too low or too high. If researchers are testing immunomodulatory agents, we recommend supplementation of the medium with IL-210.
- If using tumor tissue derived from mice, euthanize mice with a recommended procedure, sanitize the exposed skin of mice by spraying 70% ethanol, and dissect tumors with aseptic techniques. Store the tumor tissue in a tube containing ice-cold Hank’s Balanced Salt Solution (HBSS).
- If using fresh patient tumor tissues, store in ice-cold HBSS for short term, or ice-cold Belzer University of Wisconsin (UW) solution for longer term storage (up to 24 h) to preserve tissue viability.
NOTE: Slices from a PDX tissue shipped overnight in ice-cold UW solution have been successfully prepared.
- Transfer the tumor tissue into a 10 cm culture plate containing ice-cold HBSS. Cut tumor into halves with a scalpel while submerged in ice-cold HBSS. Shape tumor tissues into cylinders using a 6 mm diameter biopsy punch and trim one side to create a flat surface. Store the tissues in ice-cold HBSS until use.
NOTE: Avoid including the necrotic area of the tissue, which is generally at the center and has an opaque and fragile appearance.
- Set up the vibratome. Disinfect all the equipment using 70% ethanol. Place the vibratome buffer tray covered with an acrylic glass lid at the center of the vibratome ice bath. Add ice to the ice bath and fill up the buffer tray with cooled HBSS to keep the tissue cool while slicing. Attach a razor blade to the blade holder.
NOTE: Although keeping vibratomes in a semisterile environment has not affected prior experiments, it is recommended to store the vibratome under a biosafety cabinet if available.
- Carefully lift up a biopsy-punched tumor tissue from the HBSS using toothed forceps and remove excess buffer by dabbing the tissue with a lint-free paper towel.
- Place a drop of medical grade cyanoacrylate glue on the specimen plate and place the tissue on this drop. Air-dry the glue for 2–3 min and place the plate into the buffer tray. Supplement with some HBSS until the tissue is fully immersed in the buffer.
NOTE: Mounting tissues in an upright, stable position is critical to generate uniformly cut slices. If mounted tissues are unstable, either add more glue to the surrounding tissue to retain an upright position or unload the tissue, flatten the bottom, and glue it again. Elastic tissues tend to get pushed and bent more easily during slicing, in which case shorter lengths (<5 mm) and multiple runs may be appropriate. For extremely fragile tissues, paper towels can destroy the tissue. In this case, the tissue can be placed on a plastic surface to wick away some excess HBSS.
- Adjust the vibratome settings. The following settings are used: cutting thickness = 250 µm, amplitude = 3.00 mm, slicing speed = 0.01–0.25 mm/s, and blade angle = 15°–21°.
NOTE: Optimize the slicing settings depending on the stiffness of the tissue. Softer tissues are more easily cut with a deeper blade angle at lower speed. A blade angle of 18°, cutting speed between 0.10–0.18 mm/s, and an amplitude of 3.00 mm works well for mouse 4T1 and PDX HCI010 tumors. Stiffer tissue can be cut with a faster blade speed.
- Set the start and end locations of the blade and adjust the height of the stage. Run the instrument with continuous mode to cut slices for several cycles until the tissues are cut uniformly.
- Continue slicing the tissue. Transfer the slices along with a small amount of HBSS buffer onto the cell culture insert with medium with a wide-tip transfer pipette, using suction to lift the whole slice. Place one slice per insert on a 24 well plate. An insert for 6 well plates can accommodate up to four slices for a replicate assay.
NOTE: If the last edge of the tissue slice is still attached to the uncut tissue, stop the vibratome and separate the tissue slice with two pairs of fine tip forceps without touching or poking the tissue slices. A separate razor blade can be useful in removing hanging tissue.
- Remove any excess buffer with a fine tip transfer pipette.
- When the blade reaches close to the glued tissues, stop the instrument, lower the stage, remove the stiffened tissue with a separate blade, and mount a new tissue. Continue slicing until enough slices have been collected.
- Culture the plates in a humidified incubator set at 37 °C, 5% CO2. The tissue slices are cultured at the air-liquid interphase on the cell culture insert. The tissue slices are ready for experiments 24–48 h after the initial slice preparation. For long-term cultivation, exchange the medium every 2–3 days.
2. Viability measurement
- For viability measurement of the tissue slice, exchange the medium with a luminescence-based viability measurement reagent containing both luciferase and prosubstrate at 1:1,000 dilution in TSC medium following the manufacturer’s instructions. The reagent measures the reduction activity of live cells of metabolized prosubstrate to luciferin11. Transfer 50 µL of enzyme-substrate mixture on top of each tissue slice.
- Incubate with gentle agitation on an orbital shaker located inside a humidified incubator set at 37 °C with 5% CO2 overnight.
- Luminescent signals can be measured using either a microplate reader or an in vivo imaging instrument for tissues cultured in a 24 well plate. To measure the individual viability of multiple tissues on a 6 well plate, an in vivo imaging system should be used.
- When using a microplate reader, set the microplate without the lid. Any type of microplate reader capable of measuring luminescent intensity should be suitable for the experiment. We used the following settings: read time = 1 s, measurement from the top, gain = 200.
NOTE: For higher accuracy, use a white-wall microplate to set up the experiment. White-wall, clear-bottom 24 well plates have been tested, and in most cases, clear well plates do not compromise the results.
- When using an in vivo imaging system to measure the viability, place the plate at the center of the stage and remove the lid. Acquire normal photographic images followed by luminescent images with a field of stage setting at C, auto exposure time, f-stop = 1, objective setting as microplate with subject height = 0.0 cm.
- Quantify the luminescent intensity using the imaging software accompanied with the in vivo imaging system. Draw consistent regions of interest (ROI) around each tissue slice. This protocol uses a 1 cm diameter circle as an ROI (See Figure 1). Measure the total flux (p/s) of the area.
- When using a microplate reader, set the microplate without the lid. Any type of microplate reader capable of measuring luminescent intensity should be suitable for the experiment. We used the following settings: read time = 1 s, measurement from the top, gain = 200.
3. Evaluation of drug effect on the tissue slices
- Measure baseline viability prior to treatment using the luminescence-based viability measurement reagent as explained in section 2.
NOTE: If several tissues have significantly lower viability compared to others, omit the tissues from the assay.
- Dissolve drugs in dimethyl sulfoxide (DMSO) to make stock solutions. Prepare a 10x drug solution with TSC or other culture medium (25 µL/well for a 24 well plate containing 250 µL of medium) at the desired concentrations. For vehicle control, prepare medium containing an equivalent volume of DMSO.
- Supplement the 10x drug solution to the culture medium at the bottom of the well. Mix by pipetting and transfer 50 µL of the medium on top of the tissues.
NOTE: If several tissue slices are available, test duplicates or triplicates for each condition.
- Incubate overnight with gentle shaking on an orbital shaker in a 37 °C, 5% CO2, humidified incubator.
- Remove the plate from the shaker and incubate statically in the 37 °C, 5% CO2, humidified incubator.
- At desired timepoints, measure the luminescent intensity from the tissues using a microplate reader or an in vivo imaging system. Usually, drug effects become detectable after 1–6 days of treatment.
NOTE: The luminescence-based viability reagent is stable for at least 3 days under culture conditions. However, the substrate may be depleted during the assay depending on the metabolic activity of the tissue. If a significant signal drop is observed in tissues with previously high signals, supplement the substrate in all the wells.
- Calculate the remaining viability of the treated tumor tissues using the following equation:
The viability of the treated tissue is normalized by its baseline viability and viability shift of control tumor tissues.
Here, we demonstrate a time-course, multiple drug efficacy evaluation protocol for tumor tissue slices prepared from mouse 4T1 breast tumor tissues and a breast cancer PDX model, HCI01012. We successfully prepared tissue slices from several mouse-derived tumors, PDX, and fresh patient tumors10. The overall workflow of the tumor tissue preparation and drug efficacy test is described in Figure 1. In general, we could prepare 20–40 tissue slices from a single bulk tumor of 1,000–1,500 mm3 in volume.
For viability measurements, we exploited a luminescent-based, live cell-compatible viability reagent. Treatment of a multi-kinase inhibitor, staurosporine, at 1 µM concentration for 4 days reduced the signal intensity by 100x, compared with tumor tissues treated with DMSO control (Figure 2A). The tissue slices prepared from the 4T1 tumor were maintained for at least 21 days with occasional medium exchange (Figure 2B). Most of the drug response measurements were performed within 7 days from tissue slice preparation. Because the live cell-compatible reagent does not require processing or fixation before measurement, it allowed us to carry out time-course measurements. Time-dependent changes in the viability of tumor tissue slices from the identical 4T1 tumor tissue are summarized in Figure 2C. Staurosporine at 500 nM decreased luminescence from day 1 and reached the lowest level at day 4, remaining low for each subsequent timepoint. In contrast, the luminescent intensity from the control group of tissues supplemented with DMSO remained stable until day 6. Additionally, tissue slices prepared from identical 4T1 tumors were treated with serial doses of staurosporine (0–1 µM) for 4 days, and showed dose-dependent changes in viability and EC50 (Figure 2D).
We tested chemotherapeutic drugs on tissue slices prepared from an orthotopic PDX model of breast cancer, HCI010. PDX tissue slices were treated with doxorubicin, a standard chemotherapeutic drug, at doses of 0–5 µM for 6 days, providing dose-dependent changes in viability (Figure 3A). Further, the efficacy of 17 drugs, including preclinical and clinically approved drugs, was tested in triplicate on tissue slices prepared from a single bulk HCI010 tumor. The drugs were applied at 0.5 µM for 4 days in triplicate (Figure 3B). These results provide proof-of-concept that medium-throughput drug screening can be performed on tumor slices with native TME.
Figure 1: Schematic showing tissue slice preparation followed by drug efficacy evaluation. Tumor tissues were processed into slices 6 mm in diameter and 250 µm thick and cultured in the air-liquid interphase with support from cell culture inserts. Tissue viability was measured using a bioluminescent reagent both before and after drug treatment. Please click here to view a larger version of this figure.
Figure 2: Tissue slice viability measurement and response to the drugs. (A) Representative images of luminescence-based viability measurements in breast tumor tissue slices. Tissue slices from 4T1 tumor were incubated with 1 µM staurosporine or DMSO control together with luminescent viability assay reagent for 4 days. Images were obtained using an in vivo imaging system and analyzed by accompanying image analysis software. Red circles indicate the ROI measured for bioluminescence. Bottom = A plot showing luminescent signal from tumor tissue slices treated with staurosporine or control as measured by an in vivo imaging system. The bar indicates the mean of the four tissue slices shown as circles (control) or square (staurosporine treated). (B) A plot showing viability of 4T1 tumor tissue slices cultured for 21 days from preparation. Viability at day 21 measured by an in vivo imaging system was normalized to that of day 3. Each dot indicates measurement from a single slice; the bar indicates the mean; the box shows quartiles, and whiskers show the minimum to maximum value of the measurement. (C) Time-course measurements of tissue viability after the staurosporine treatment. The viability of tissue slices from the 4T1 tumor treated with staurosporine (500 nM) or equivalent volume of DMSO as a control were measured over time. Luminescence intensities were measured by a microplate reader over the indicated timepoints. Each dot illustrates a datapoint from an individual tissue slice, and the bar graph indicates mean ± SEM. (D) Dose-dependent treatment of staurosporine on tissue slices. Tissue slices prepared from 4T1 tumor were supplemented with serial doses of staurosporine or DMSO as a vehicle control. The remaining viability based on luminescence was measured by an in vivo imaging system at 4 days after treatment initiation. Relative viability was calculated using the equation shown in the protocol. Please click here to view a larger version of this figure.
Figure 3: Drug screen in tissue slices prepared from a breast cancer PDX model. (A) A plot showing changes in viability in response to various doses of doxorubicin. The tissue slices were prepared from an orthotopic breast cancer PDX tumor, HCI010. Viability was measured in slices treated with titrated doses of doxorubicin. After 6 days, luminescence was measured using an in vivo imaging system. Bar = mean ± SEM. (B) A small-scale chemotherapeutic drug screen on tumor tissue slices from an orthotopic breast cancer PDX tumor. The bar graph = mean ± SEM of triplicate experiment. These data have been published previously10. Please click here to view a larger version of this figure.
|William's Medium E||500|
|Nicotinamide||6||12 mM||1 M stock of Nicotinamide in Williams’ Medium E, sterilized|
|Ascorbic Acid||6||50.4 μg/mL||0.21 g/50 mL (>10 mM stock) of Ascorbic Acid in William's Medium E, sterilized|
|Sodium Bicarbonate||15||2.25 mg/mL||7.5% (w/v) solution|
|HEPES Buffer||10||20 mM||1 M stock solution|
|Glucose||10||5 mg/mL||250 mg/ml stock, steralized|
|Sodium Pyruvate||5||1 mM||100 mM stock|
|L-Glutamine||5||2 mM||200 mM stock|
|ITS + Premix||5||Contains human recombinant insulin, human transferrin (12.5 mg each), selenous acid (12.5 µg), BSA (2.5 g), and linoleic acid (10.7 mg)|
|Penicillin-Streptomycin||2||40 IU/mL Pen, 40 μg/mL Strep||10,000 IU/mL Pen, 10,000 μg/mL Strep|
|Recombinant EGF||0.5||20 ng/mL||20 µg/mL stock|
Table 1: TSC medium recipe.
In this protocol, we demonstrate a platform for quantitative, and real-time drug efficacy studies on organotypic tumor tissue slices. The tissue slice culture system provides distinct advantages over traditional cell-based in vitro methods by capturing cell heterogeneity and physiological characteristics of the native tumor microenvironment. This platform also enables higher throughput for drug efficacy testing, helping to bridge the gap between cell culture studies and in vivo experiments.
To obtain accurate and consistent results, it is imperative to minimize tissue damage during preparation. Tissue slices should be handled with wide tip plastic pipettes to avoid damage caused by physical contact. The culture medium volume must be maintained to allow the tissue slice to contact both the air and medium for the duration of the experiment.
The culture medium should be optimized depending on the tissue type and experimental purpose. The TSC medium used in this protocol is a serum-free medium that was originally developed for primary hepatocyte culture13. This medium has also been used to maintain several types of tumors, including breast, liver, colon, and pancreas10. Further, we demonstrated enhanced immune cell survival using an optimized DMEM-based medium10. Vibratome settings should be optimized based on tissue texture to obtain intact and uniform tumor tissue slices. Slicing softer and/or loosely consolidated tissues is typically made easier by using deeper blade angles, slower cutting speed, and thicker slices. Occasionally, tissues are too soft to cut, in which case researchers should consider embedding the tissue within low melting point agarose, a technique common in brain tissue slicing14,15.
Previously, several studies introduced therapeutic drug testing approaches on cultured tissue slices. These studies relied on fluorescent dye incorporation, immunohistochemistry, or MTT assays to evaluate tissue viability6,7,8,9. In this protocol, we used the luminescence-based, live cell-compatible reagent RealTime-Glo11 for viability measurement. Luminescence-based assays have several benefits over previously used methods, including increased sensitivity, wider signal range, and the ability to rapidly make real-time viability measurements. Measuring time is short for both microplate readers (~1 s/well) and an in vivo imaging system (~1 min/plate) and can provide signal intensity readings directly with minimal processing. Faster acquisition of the signal and fewer postprocessing steps are necessary features in high-throughput drug screening, allowing luciferin-based viability measurements to enable a much greater sample throughput in tissue slice culture. While a luciferase-based assay provides an accurate, quantitative measurement of the whole slice viability, it is unable to detect drug-induced cell type-specific changes in the tissue. However, coupling luminescence-based viability measurements with end point IHC on the same tissue slice will enable cell type-specific measurements.
While the tissue slice culture system is a powerful tool in bringing tumor tissue complexity to a high-throughput screening platform, it has several drawbacks. Inherent heterogeneity within a tumor tissue may cause differential drug responses among tissue slices even from the same bulk tumor. On occasion, we observed relatively large variations in drug response among tissue slices in our experiments. We can partially overcome this variation by averaging multiple tissue slices prepared from different sites within the same tumor. Although tissue slice culture can be maintained at baseline level of viability for at least 21 days (Figure 2B), cell populations change over time. We have described shifts in immune cell population during the course of tissue slice culture experiments10. Therefore, we recommend tissue slices be examined over shorter culturing time intervals to recapitulate native tissue characteristics.
Tissue slice culture is poised to fill a critical gap in drug discovery and development between high throughput cell culture screening and animal experiments. Hundreds of drugs can be evaluated on slices produced from a single tumor biopsy, adding increased physiological relevance prior to animal studies. This system could help to reduce the number of animal experiments required to bring drugs to market. Furthermore, tissue slices prepared from patient tumors have informative potential in guiding treatment plans in the clinic. Over a hundred therapeutic drugs are available today, yet there are few biomarkers to guide their selection. Moreover, heterogeneity in patients also results in disparities in response. Tissue slices from a patient biopsy can be used to test multiple drugs before treating systemically, providing valuable information on drug efficacy within a week’s time. Overall, real-time and rapid testing of drugs using tissue slice culture systems has great potential in cancer drug development and therapeutic decisions.
The authors have nothing to disclose.
This work was supported by the NIH/NCI [K22CA201229, P30CA015704], and Sidney Kimmel Foundation [Kimmel Scholar Award], Lung Cancer Discovery Award (LCD-505536). We would like to thank Dr. Alana Welm (University of Utah) for providing the breast cancer PDX tumor. We would also like to thank the staff at Comparative Medicine, Fred Hutchinson Cancer Research Center (FHCRC) for maintenance of the PDX model and members of the Gujral lab for helpful discussions. N.N.A is supported by the JSPS Overseas Research Fellowship and Interdisciplinary Training Grant from FHCRC. A.J.B. is supported by the Chromosome Metabolism and Cancer Training Grant from FHCRC.
|10 cm dish||Corning||430293|
|6 mm diameter Biopsy punch||Integra Miltex||33-36|
|Ascorbic Acid||Sigma-Aldrich||A8960-5g||For TSC medium|
|Belzer UW cold storage solcution (UW solution)||Bridge to Life||500 mL|
|Corning Matrigel Membrane Mix||Fisher scientific||356234|
|Double Edge Stainless Steel Cutting Blades||TED PELLA||121-6||For slicing|
|Doxorubicin (hydrochloride)||Cayman Chemical||15007|
|Fine tip forceps||ROBOZ||RS-4974|
|Fine tip forceps||ROBOZ||RS-4976|
|Glucose||Sigma-Aldrich||G5767-500g||For TSC medium|
|HBSS without Calcium, Magnesium or Phenol Red||Gibco||14-175-103|
|HCI010||Dr. Alana Welm, University of Utah||Breast cancer PDX|
|HEPES Buffer Solution||Gibco||15630080||For TSC medium|
|ITS + Premix||Fisher Scientific||354352||For TSC medium|
|Leica VT1200S Vibratome||Leica||VT1200S|
|L-Glutamine||Gibco||25030164||For TSC medium|
|Millicell Cell Culture Insert, 12 mm, hydrophilic PTFE, 0.4 µm||Millipore||PICM01250|
|Millicell Cell Culture Insert, 30 mm, hydrophilic PTFE, 0.4 µm||Millipore||PICM03050|
|Nicotinamide||Sigma-Aldrich||N0636-500g||For TSC medium|
|PELCO Pro CA44 Tissue Adhesive||TED PELLA||10033||Glue|
|Penicillin-Streptomycin||Fisher Scientific||15140163||For TSC medium|
|RealTime-Glo MT Cell Viability Assay||Promega||G9711|
|Recombinant Mouse EGF||BioLegend||585608||For TSC medium|
|Single Edge Industrial Razor Blades||VWR||55411-050||For removing glued tissues|
|Sodium Bicarbonate||Corning||25-035-CI||For TSC medium|
|Sodium Pyruvate||Fisher Scientific||BW13115E||For TSC medium|
|Staurosporine||Santa Cruz Biotechnology||sc-3510A|
|Transfer pipettes||Fisher scientific||13-711-7M||Wide tip|
|Transfer pipettes||Samco Scientific||235||Fine tip|
|William's medium E, no glutamine||ThermoFisher Scientific||12551032||For TSC medium|
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