Isolation, In Vitro Expansion, and Characterization of Mesenchymal Stem Cells from Mouse Epididymal Adipose Tissue

Summary

The adipose tissue is an excellent source of mesenchymal stem cells. Here, we bring the step-by-step extraction, cultivation, and characterization of adipose tissue-derived stem cells (ADSCs) from Swiss mice epididymal adipose tissue.

Abstract

Mesenchymal stem cells (MSCs) have been extensively studied as a new therapeutic approach, mainly to stop exacerbated inflammation due to their potential to modulate the immune response. The MSCs are immune-privileged cells capable of surviving in immunologically incompatible allogeneic transplant recipients based on low expression of class I major histocompatibility complex (MHC) molecules and in the use of cell-based therapy for allogeneic transplant. These cells can be isolated from several tissues, the most commonly used being the bone marrow and adipose tissues. We provide an easy protocol to isolate, culture, and characterize MSCs from epididymal adipose tissue of mice. The epididymal adipose tissue is surgically excised, physically fragmented, and digested with 0.15% collagenase type II solution. Then, primary adipose tissue-derived stem (ADSCs) cells are cultured and expanded in vitro, and the phenotypic characterization is performed by flow cytometry. We also provide the steps to differentiate the ADSCs into osteogenic, adipogenic, and chondrogenic cells, followed by functional characterization of each cell lineage. The protocol provided here can be used for in vivo and ex vivo experiments, and as an alternative, the adipose-derived stem cells can be used to generate MSCs-like immortalized cells.

Introduction

Mesenchymal stem cells (MSCs) are adult multipotential cells differentiating into cells such as osteoblast, chondroblast, and adipocyte^1,2. These cells reside in several organs, and because of that, they can be extracted from adult tissues such as bone marrow, muscle, fat, hair follicle, tooth root, placenta, dermis, perichondrium, umbilical cord, lung, liver, and spleen^3,4.

The effects of MSCs on physiology and the immune system have been reported^5,6. These cells have been promising for treating several diseases, both in human and veterinary medicine. The MSCs can control inflammation and promote angiogenesis and tissue homeostasis through different mechanisms, such as cell-cell contact, soluble factors, and small extracellular vesicles^7,8,9,10. Furthermore, the MSCs are immune-privileged cells capable of surviving in immunologically incompatible allogeneic transplant recipients because these cells show low expression of class I major histocompatibility complex (MHC) molecules and are used in cell-based therapy for allogeneic transplant^11,12. The low immunogenicity combined with the regenerative potential makes MSCs ideal candidates for cell therapy, such as graft-versus-host disease (GvHD)¹³, systemic lupus erythematosus (SLE)¹⁴, and multiple sclerosis¹⁵, among others^16,17.

Despite the fact that MSCs reside in several adult tissues, the adipose tissue offers advantages over other sources, such as accessibility for harvesting, with minimal surgical intervention; large number of available cells with high expansion rate; and easy in vitro expansion using an easy-to-perform protocol without the need for specific equipment and low-cost materials^18,19,20. Once extracted, the adipose tissue-derived stem cells (ADSCs) must be characterized as established by the International Society for Cellular Therapy (ISCT)²¹. Thus, MSCs must show morphology fibroblast-like, adherence to plastic culture, expressing a high percentage (≥95%) of mesenchymal markers such as endoglin (CD105), ecto-5'-nucleotidase (CD73) and Thy-1 (CD90), and low percentage (≤2%) of hematopoietic markers such as leukocyte common antigen (CD45), transmembrane phosphoglycoprotein (CD34), glycolipid-anchored membrane glycoprotein (CD14), integrin alpha M (CD11b), B-cell antigen receptor complex-associated protein alpha chain (CD79α) or B-lymphocyte surface antigen B4 (CD19) and class II human leukocyte antigen (HLA-II). Furthermore, a functional characterization is required, and the cells should be able to differentiate into osteoblast, chondroblast, or adipoblast cells²¹.

Here, we show how to obtain the MSCs from epididymal adipose tissue using mechanical dissociation and enzymatic digestion for in vitro studies and the morphological characterization preconized by ISCT.

Protocol

All animal experiments were conducted according to international guidelines for animal ethics and were approved by institutional committees of care and use from the State University of Santa Cruz under protocol number 021/22. Swiss male mice (6-8 weeks) were acquired from the Animal Breeding, Maintenance and Experimentation Laboratory - State University of Santa Cruz (LaBIO-UESC) Animal Research Facility, maintained in specific pathogen-free conditions, receiving water and food ad libitum with 12 h light/dark cycles.

NOTE: Other mice lineage can be used; we recommend the use of adult mice (6-8 weeks) since they have more developed adipose tissue and cells with high proliferative activity.

1. Preparation

NOTE: Before proceeding, prepare the following reagents described below. See the Table of Materials for reagent and material supplier information. Personal protective equipment such as masks, caps, lab coats, and gloves must be worn in all practical procedures.

1. Epididymal adipose tissue extraction
   1. Reserve the surgery materials: three sets of sterile tweezers and scissors, five needles, and a beaker containing 200 mL of 70% ethanol.
   2. Prepare a terminal anesthetic, mixing ketamine (100 mg/kg) and xylazine (10 mg/kg).
2. ADSCs culture
   1. Basal medium: Prepare Dulbecco's Modified Eagle Medium (DMEM) high glucose media supplemented with 10% fetal bovine serum (FBS), 50 µg/mL gentamicin, 0.25 µg/mL amphotericin B, 100 U/mL penicillin, and 100 mg/mL streptomycin.
   2. Digest solution: Prepare 0.15% of collagenase type II in PBS sterilized in a 0.25 µm filter.
      NOTE: It is not necessary to use the four antibiotics described in the basal medium. It will depend on the cell culture conditions of the laboratory where this is performed.
3. ADSCs phenotypic characterization
   1. Prepare 1% bovine serum albumin (BSA) in PBS.
   2. Dilute the anti-mouse antibodies as mentioned in Table 1.
   3. Prepare 2% formaldehyde in PBS.
4. ADSCs osteogenic differentiation
   1. Osteogenic differentiation medium: Prepare DMEM supplemented with 5.67 M ascorbic acid, 10 nM dexamethasone, 0.02 M β-glycerophosphate, 10% FBS, and 1% penicillin/streptomycin.
   2. For Von Kossa staining, prepare the following solutions: 70% ethanol in water, 5% silver nitrate in water, distilled water, 5% sodium thiosulfate in water, and 1% eosin B in water.
5. Adipogenic differentiation:
   1. Adipogenic differentiation medium: Prepare DMEM supplemented with 0.5 mM 3-Isobutyl-1-methylxanthine, 200 µM indomethacin, 1 µM dexamethasone, 10 µM insulin, 10% FBS, and 1 % penicillin/streptomycin.
   2. For Oil-red staining, prepare the following solutions: 10% formalin in deionized water, 60% isopropanol in deionized water, lysochrome (fat-soluble dye) diazo dye (Oil-Red O solution) in 60% isopropanol, and 1% hematoxylin in deionized water.
6. Chondrogenic differentiation:
   1. Chondrogenic differentiation medium: Prepare chondrogenic medium supplemented with 10% FBS and 1% penicillin/streptomycin.
   2. Prepare 10% formaldehyde in PBS.
   3. For proteoglycans and glycosaminoglycans staining, prepare the following solutions: Heteroglycan stain (Alcian Blue 1%) in acetic acid, pH 2.5, paraffin, hematoxylin, ethanol series dilution in water (100%, 96%, 80%, and 70%).

Antibody/Fluorophore	Dilution	Clone	Final concentration (µg/mL)	Function and cell in which it is expressed
CD34- PE	1:100	RAM34	2	Cell adhesion factor. Hematopoietic stem cells.
CD45- APC	1:100	30-F11	2	Assistence in activation of leukocytes. Expressed in leukocytes.
CD71- FITC	1:100	C2	5	Controls iron uptake during cell proliferation. Proliferating cells, reticulocytes, and precursor cells.
CD29- FITC	1:100	Ha2/5	5	Adhesion and activation, embryogenesis, Leukocytes, dendritic cells, platelets, mast cells, fibroblasts, and endothelial cells.
CD90- PerCP	1:100	OX-7	2	Signaling, adhesion. T lymphocyte, NK, monocyte, Hemtopoietic Stem Cells, neuron, and fibroblast.

Table 1: Antibodies used for phenotypic characterization of ADSCs by flow cytometer. List of antibodies with their respective fluorochromes, dilutions, clone, and final concentration as well as their function in the cell which is expressed.

2. Methods

NOTE: The adipose tissue is distributed throughout the body in subcutaneous or intra-abdominal locations. In mice, the most common adipose tissues used for experiments include the subcutaneous epididymal, mesenteric, and retroperitoneal²². Here, the steps for obtaining epididymal adipose tissue are shown (Figure 1).

Figure 1: Experimental design to obtain adipose tissue-derived stem cells from Swiss mice. (A) Using needles, place the animal on the cork or styrofoam board; (B) Lift the skin using tweezers, make a cut in the center of the abdominal region, and detach the skin from the peritoneum; (C) identify the white adipose tissue above the epididymis; (D) transfer the tissue to DMEM medium supplemented with 10% FBS and 1% antibiotics, wash the epididymal adipose tissue thoroughly with PBS, and transfer the tissue to digest solution; (E), fragment the tissue and (F) incubate at 37 °C for 1 h vigorously shaking every 10 min; (G) after counting the cells, plate in 6-well plates and observe the cell morphology under an inverted microscope. (F) Cell morphology will change from round to fibroblast-like. Scale bar = 22.22 µm. Please click here to view a larger version of this figure.

1. Extraction of epididymal adipose tissue
   NOTE: Be aware that all procedures must be carried out under sterile conditions in a laminar flow cabinet in order to ensure pure and contamination-free cell culture.
   1. Euthanize the animals using ketamine and xylazine solution (100 and 10 mg/kg, as described in step 1.1.2) by intraperitoneal route.
      NOTE: Other euthanizing methods can be used²³. Make sure the animal is dead by confirming the absence of a heartbeat.
   2. Place the animal with the ventral side facing up on a cork or styrofoam board covered with aluminum foil and fix it using sterile needles (Figure 1A).
      NOTE: To avoid contamination, spray 70% alcohol in the abdominal region. Alternatively, shave the hair with a scalpel.
   3. Lift the skin using tweezers in the center of the abdominal region and make a cut with sterile scissors.
      NOTE: Two sets of sterile tweezers and scissors are necessary to avoid contamination. The first one is used to open the animal, cutting the skin and peritoneal layer; the second one is used to pick up the epididymal adipose tissue. Also, reserve 150 mL of 70% alcohol in a beaker to clean scissors and tweezers between steps.
   4. Introduce the scissors under the animal's skin and open it to detach it from the peritoneum (Figure 1B). Lift the peritoneal layer using tweezers and cut with sterile scissors.
   5. Use another set of sterile tweezers and scissors and identify the epididymis above the testes and the white adipose tissue above the epididymis. Pull the entire epididymal adipose tissue using sterile tweezers, approximately 2 cm in size, and cut very close to the epididymis (Figure 1C).
   6. Put the fat into a 50 mL sterile conical tube containing basal medium and place it on ice (Figure 1D). In the hood, proceed to the next steps as described below.
2. Adipose tissue-derived stem cells (ADSCs) isolation
   NOTE: Process the samples in a biological class II laminar flow hood, and personnel should wear a lab coat, gloves, and surgical mask.
   1. Wash the epididymal adipose tissue thoroughly with PBS.
      NOTE: This step is essential to remove the blood and other particles from the sample. The blood can inhibit collagenase type II enzyme and compromise the experiment.
   2. Using a sterile tweezer, transfer the epididymal adipose tissue into a 50 mL tube containing 15-20 mL of digest solution, prepared as step 1.2.2. Fragment the tissue using sterile scissors and incubate the tissue at 37 °C for 1 h (Figure 1E, F).
      NOTE: A water bath or incubator at 37 °C can be used in this step. Every 10 min, the suspension must be vigorously shaken to facilitate digestion. Do not extend the time of 1h incubation.
   3. Inactivate the collagenase by diluting the sample 1:1 in basal medium and centrifuge the suspension at 250 x g for 10 min at 4 °C to obtain the vascular fraction of the stroma. Discard the supernatant and resuspend the pellet in 1 mL of basal medium.
   4. Check the viability and count the cells through the trypan blue dye exclusion method in a Neubauer chamber using a dilution of 1:10 or 1:100.
      NOTE: The expected yield is around 0.5-1 million cells/g of processed adipose tissue and viability of 80%.
   5. Plate the isolated cells (0.3-0.5 million) in 3 mL of basal medium in one well of a 6-well plate. Incubate the cells overnight at 37 °C with 5 % CO₂.
   6. Next day: Remove the medium from the well and wash the cells with PBS. Add 3 mL of basal medium. Repeat this process every 2 days until the cells become 90% confluent.
      NOTE: Always observe the morphology of the cells under the inverted microscope, changing from round to fibroblast-like (Figure 1H).
3. Adipose tissue-derived stem cells (ADSCs) expansion
   NOTE: Once the cells grow ~90% confluent, proceed to subculture as described below.
   1. Remove the medium from the well and wash the cells with PBS. Add 0.5 mL/well of trypsin/EDTA (0.05 % trypsin; 200 mg/L EDTA) and incubate the plate for 3-5 min at 37 °C to detach the cells.
      NOTE: Do not exceed 5 min in trypsin/EDTA. The complete detachment of the cells should be verified under the inverted microscope. The process can be accelerated by pipetting up and down carefully or tapping the side of the plate.
   2. Inactivate the enzyme by diluting the sample 1:1 in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics.
   3. Split the cell suspension into 2 wells and add 3 mL of basal medium (DMEM supplemented with 10 % FBS and 1 % antibiotics). Incubate overnight at 37 °C with 5% CO₂.
   4. Change medium the next day, then every 2 days until 90% confluence.
   5. Repeat the process until the third passage and proceed to phenotype and functional characterization.
      NOTE: Always observe the morphology of the cells under the inverted microscope, changing from round to fibroblast-like.
4. Adipose tissue-derived stem cells (ADSCs) characterization
   1. Phenotypic characterization by flow cytometry
      1. Detach the cells using Trypsin/EDTA, as described in steps 2.3.1 to 2.3.2.
      2. Collect the cells in a conical tube (50 mL) and centrifuge at 250 x g for 10 min at 4 °C. Discard the supernatant and resuspend the pellet in 1 mL of PBS.
      3. Count the cells as described in step 2.2.4 and seed 0.5-1 x 10⁶ cells in 100 µL of PBS in a 96 well-plate.
         NOTE: The number of wells will depend on the color of the fluorochrome conjugate and the filters/lasers of the cytometer.
      4. Add antibodies (Table 1) diluted in 1% BSA in PBS.
         NOTE: It is not necessary to use Fc block to avoid nonspecific binding because we already use BSA to dilute antibodies. Reserve a sample of cells without receiving antibodies that will be used as a control of staining. The antibodies can be combined in the same well according to the fluorochrome dye and cytometer available in the lab. All the antibodies described in Table 1 can be added in the same well, except the CD71 FITC and CD29 FITC, which must be in different wells because of the same fluorochrome.
      5. Incubate the plate for 30 min at 4 °C in the dark.
      6. Wash cells, then complete the volume to 200 µL using PBS, centrifuge the plate at 252 x g for 10 min at 4 °C, and discard the supernatant. Repeat this step one more time.
      7. Fix the cells by adding 200 µL per well of 2% formaldehyde in PBS. Store the cells in the dark at 4 °C until analysis in the flow cytometer.
         NOTE: For best results, acquire the cells on the flow cytometer as soon as possible. The brightness of fluorescence of markers decays significantly after 48 h for most fluorochromes. So, do not exceed 48 h to read the plate. Acquisition of 30,000 events is recommended.
      8. Use the gating strategy presented in Figure 2B to select the ASC population.
   2. Functional characterization: Osteogenic differentiation
      1. Detach the cells using Trypsin/EDTA (as described in steps 2.3.1 to 2.3.2), collect and centrifuge the cells (as described in steps 2.4.1.2 and 2.4.1.2), and count them (as described in steps 2.2.4).
      2. Plate, in triplicate, 2 x 10⁵ cells per well in 6-well plates in basal medial (DMEM supplemented with 10 % FBS and 1 % antibiotics); incubate at 37 °C in 5% CO₂.
         NOTE: Two 6-well plates will be needed, one for each differentiation time (14 and 21 days).
      3. Next day, change the medium to osteogenic medium (step 1.4.1).
         NOTE: reserve 3 wells being cultured only with the basal medium, which will be the control for differentiation.
      4. Culture cells for 14 and 21 days in osteogenic media and the control cells, changing media every 3 days. Proceed to Von Kossa staining (step 1.4.2) to assess the mineralized nodules at the end of each culture period.
      5. Aspirate the medium from each well and wash the cells twice with PBS. Fix the cells with 2 mL of 70% ethanol overnight at room temperature (RT). Wash the cells carefully in running water.
      6. Add 2 mL of 5% silver nitrate solution and incubate the plate in ultraviolet light for 1 h. Wash the cells with distilled water. Add 2 mL of 5% sodium thiosulfate, and incubate for 5 min. Wash with distilled water.
      7. Counterstain with 2 mL of eosin for 40 s. Wash with distilled water until the staining solution is removed, and visualize the staining using an optical microscope.
   3. Functional characterization: Adipogenic differentiation
      1. Detach the cells using Trypsin/EDTA (as described in steps 2.3.1 to 2.3.2), collect and centrifuge the cells (as described in steps 2.4.1.2 and 2.4.1.2), and count them (as described in steps 2.2.4).
      2. Plate 2 x 10⁵ cells per well in 6-well plates in basal medial (DMEM supplemented with 10 % FBS and 1 % antibiotics); incubate at 37 °C in 5% CO₂.
         NOTE: Two 6-well plates will be needed, one for each differentiation time (14 and 21 days).
      3. The next day, change the medium to adipogenic medium (step 2.5.1).
         NOTE: Reserve 3 wells being cultured only with the basal medium as control.
      4. Culture cells for 14 and 21 days in adipogenic medium, changing media every 3 days. At the end of each culture period, proceed to Oil-Red O staining (steps 2.4.3.5-2.4.3.7), an indicator of intracellular lipid accumulation.
      5. Aspirate the media from each well. Wash the cells twice with PBS. Fix cells in 2 mL of 10% formalin for 1 h. Wash once with PBS, and then one time with water.
      6. Stain with 2 mL of Oil-Red O solution in 60% isopropanol for 5 min. Wash once with distilled water.
      7. Counterstain with 2 mL of 1% hematoxylin diluted 1:2 in distilled water for 1 min. Wash with distilled water until the staining solution is removed, and visualize the stained samples using an optical microscope.
   4. Functional characterization: Chondrogenic differentiation
      1. Detach the cells using Trypsin/EDTA (as described in steps 2.3.1 to 2.3.2), collect and centrifuge the cells (as described in steps 2.4.1.2 and 2.4.1.2), and count them (as described in steps 2.2.4).
      2. Place 5 x 10⁵ ADSCs in four polypropylene conical tubes and centrifuge at 450 x g for 5 min to form a pellet. Discard the supernatant.
      3. Culture the pellets in a conical tube in a chondrogenic medium (step 1.6.1) or only basal medium (control of differentiation) for 14 and 21 days at 37 °C in 5% CO₂. Change medium every 3 days and avoid removing pellets.
         NOTE: Each cell pellet refers to each culture time, as well as their respective controls grown only with basal medium.
      4. At the end of each culture time point, collect pellets using fine-tip tweezers and place them in a 24-well plate. Proceed for histological sectioning and staining of proteoglycans and glycosaminoglycans (steps 2.4.4.5.-2.4.4.9).
         NOTE: Each pellet is processed in a separate well.
      5. Fix pellets in 2 mL of 10% buffered formaldehyde for 30 min. Discard the formaldehyde solution, and add 2 mL of 70% ethanol. Remove the pellet from the conical tube using a tip and embed the pellets in paraffin.
      6. Using a rotating paraffin microtome, make 5 µm histological sections. Incubate the sections for 15 min at 60 °C, and immerse them twice in xylene (for 5 and 15 min).
      7. Rehydrate the samples by immersing the histological sections in alcohol baths at decreasing concentrations (100%, 96%, 80%, and 70%) for 2 min each, then rinse with deionized water for 5 min.
      8. Proceed to stain the samples with Alcian blue 1% in acetic acid, pH 2.5, for 30 min.
      9. Counterstain with hematoxylin for 1 min, and wash with distilled water. Mount with DPX (mountant for histology) or other mount solution and visualize samples using an optical microscope.

Results

Cells extracted from adipose tissue according to the protocol presented here showed morphology matching the minimal criteria for MSCs proposed by ISCT. An overview of the protocol is shown in Figure 1. Phenotypically, ADSCs showed adherence to plastic and fibroblast-like morphology in the first days of cell culture (Figure 2A). In addition, they grew homogeneously and formed colonies. Furthermore, ADSCs showed low expression of CD34 (2.12%) and CD45 (1.81%), bot...

Discussion

The MSCs can be extracted from different tissues. Despite bone marrow representing a common source of MSCs in both murine and humans^25,26, we have chosen to work with adipose tissue in this study because of its richness in MSCs, distribution in the body, and ease of accessing it. As an alternative, adipose-derived stem cells can be used to generate MSCs-like immortalized cells²⁷.

Some points of the extraction d...

Materials

Name	Company	Catalog Number	Comments
140 °C High Heat Sterilization CO2 Incubator	RADOBIO SCIENTIFIC CO. LTD, China	C180
3-Isobutyl-1-methylxanthine	Sigma-Aldrich, San Luis, Missouri, USA	I7018
Acetic acid glacial	Sigma-Aldrich, San Luis, Missouri, USA	PHR1748
Alcian Blue 8GX	Sigma-Aldrich, San Luis, Missouri, USA	A9186	BioReagent, suitable for detection of glycoproteins. 1% in acetic acid, pH 2.5
Alcohol 70%	Sigma-Aldrich, San Luis, Missouri, USA	65350-M	70% in water
Amphotericin B	Sigma-Aldrich, San Luis, Missouri, USA	PHR1662
Antibodies anti-mouse anti-CD29 FITC (Clone Ha2/5)	BD Biosciences, San Diego, CA, USA	555005	Functions in the cell: Adhesion and activation, embryogenesis, Leukocytes, DC, platelets, mast cells, fibroblasts and endothelial cells
Antibodies anti-mouse anti-CD34 PE (Clone RAM34)	BD Biosciences, San Diego, CA, USA	551387	Functions in the cell: Cell adhesion factor. Hematopoietic stem cells
Antibodies anti-mouse anti-CD45 APC (Clone 30-F11)	BD Biosciences, San Diego, CA, USA	559864	Functions in the cell: Assists in the activation of leukocytes
Antibodies anti-mouse anti-CD71 FITC (Clone C2)	BD Biosciences, San Diego, CA, USA	553266	Functions in the cell: Controls iron uptake during cell proliferation. Proliferating cells, reticulocytes and precursors
Antibodies anti-mouse anti-CD90 PerCP (Clone OX-7)	BD Biosciences, San Diego, CA, USA	557266	Functions in the cell: Signaling, adhesion. T lymphocyte, NK, monocyte, HSC, neuron, fibroblast
Ascorbic acid	Sigma-Aldrich, San Luis, Missouri, USA	PHR1008
Automatic pipettes	Thermo Fisher Scientific, Waltham, Massachusetts, USA	4700850N	Finnpipette F1 Good Laboratory Pipetting (GLP) Kits
Beaker	Not applicable		1 unit
Bovine serum albumin	Sigma-Aldrich, San Luis, Missouri, USA	A7906
Cell culture plates (6-well)	Merck, Darmstadt, Germany	Z707759	07 units sterile. TPP tissue culture plates
Cell culture plates (96-well. Round or V bottom)	Merck, Darmstadt, Germany	CLS353077	01 unit sterile. Wells, 96, Tissue Culture (TC)-treated surface, round bottom clear wells, sterile
Chondrogenic medium	Stem Pro Chondrogenesis Differentiation–Life Technologies	A1007101	TGF-β2, TGF-β3, dexamethasone, insulin, transferrin, ITS, sodium-l - ascorbate, sodium pyruvate, ascorbate-2-phosphate
Collagenase type II	Life Technologies, California, USA	17101015
cork or styrofoam board covered with aluminum	Not applicable		1 unit
cotton	Not applicable		50 g
Dexamethasone	Sigma-Aldrich, San Luis, Missouri, USA	D4902
Dissecting scissor	Not applicable		03 units sterile
DPX Mountant for histology	Sigma-Aldrich, San Luis, Missouri, USA	6522
Dulbecco’s modified Eagle’s medium (DMEM)	Sigma-Aldrich, San Luis, Missouri, USA	D5523	With 1000 mg/L glucose and L-glutamine, without sodium bicarbonate, powder, suitable for cell culture
Eosin B	Sigma-Aldrich, San Luis, Missouri, USA	861006
Fetal bovine serum (FBS)	Sigma-Aldrich, San Luis, Missouri, USA	F4135
Formaldehyde	Sigma-Aldrich, San Luis, Missouri, USA	47608
Formalin	Sigma-Aldrich, San Luis, Missouri, USA	HT501128
Gentamicin	Sigma-Aldrich, San Luis, Missouri, USA	G1397
Hematoxylin	Sigma-Aldrich, San Luis, Missouri, USA	H3136
Hypodermic Needle (0.3mm x 13mm)	Not applicable		5 units
Indomethacin	Sigma-Aldrich, San Luis, Missouri, USA	I0200000
Insulin	Sigma-Aldrich, San Luis, Missouri, USA	I3536
Isopropanol	Sigma-Aldrich, San Luis, Missouri, USA	563935	70% in H2O
Ketamine-D4 hydrochloride solution	Sigma-Aldrich, San Luis, Missouri, USA	K-006	1.0 mg/mL in methanol (as free base), certified reference material, Cerilliant®
Neubauer chamber	Sigma-Aldrich, San Luis, Missouri, USA	BR718620	BRAND counting chamber BLAUBRAND Neubauer pattern. With clips, double ruled
Nichiryo pipette tips (0.1–10 μL)	Merck, Darmstadt, Germany	Z645540	Volume range 0.1–10 μL, elongated, bulk pack. Sterile
Nichiryo pipette tips (1–10 mL)	Merck, Darmstadt, Germany	Z717401	Volume range 1–10 mL, universal, bulk pack. Sterile
Nichiryo pipette tips (200 μL)	Merck, Darmstadt, Germany	Z645516	Maximum volume 200 μL, graduated, ministack. Sterile
Oil-Red O solution	Sigma-Aldrich, San Luis, Missouri, USA	O1391	0.5% in isopropanol
Paraffin	Sigma-Aldrich, San Luis, Missouri, USA	107.151	46–48, in block form
Penicillin/Streptomycin	Sigma-Aldrich, San Luis, Missouri, USA	P4333	Solution stabilized, with 10,000 units penicillin and 10 mg streptomycin/mL, 0.1 μm filtered, BioReagent, suitable for cell culture
Phosphate-buffered saline solution 1x (PBS).	Sigma-Aldrich, San Luis, Missouri, USA	P3813	Powder, pH 7.4, for preparing 1 L solutions. Balanced and sterile
Polypropylene conical tubes (15 mL)	Falcon, Fisher Scientific	14-959-53A	Sterile
Polypropylene conical tubes (50 mL)	Falcon, Fisher Scientific	14-432-22	2 units sterile
scalpel (optional)	Not applicable		1 unit
Silver nitrate	Sigma-Aldrich, San Luis, Missouri, USA	85228
Sodium thiosulfate	Sigma-Aldrich, San Luis, Missouri, USA	72049
Surgical tweezer (15 cm)	Not applicable		3 units sterile
Swiss male mice (6–8 weeks)	Bioterium, Santa Cruz State University	021/22
syringe (1 mL)	Not applicable		1 unit
Trypan Blue Dye	Sigma-Aldrich, San Luis, Missouri, USA	T8154	0.4%, liquid, sterile-filtered, suitable for cell culture
Trypsin/EDTA (ethylenediaminetetraacetic acid)	Sigma-Aldrich, San Luis, Missouri, USA	T3924
Xylazine	Sigma-Aldrich, San Luis, Missouri, USA	PHR3263
β-glycerophosphate disodium salt hydrate	Sigma-Aldrich, San Luis, Missouri, USA	G9422	BioUltra, suitable for cell culture, suitable for plant cell culture, ≥99% (titration)