Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell (MSC) Spheroids Primed in 3-D Cultures Under Xeno-free Conditions

Summary

The therapeutic potential of mesenchymal stem/stromal cells (MSCs) is well-documented, however the best method of preparing the cells for patients remains controversial. Herein, we communicate protocols to efficiently generate and administer therapeutic spherical aggregates or 'spheroids' of MSCs primed under xeno-free conditions for experimental and clinical applications.

Abstract

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via their strong adherence to tissue culture plastic and then further expanded in vitro, most commonly using fetal bovine serum (FBS). Since FBS can cause MSCs to become immunogenic, its presence in MSC cultures limits both clinical and experimental applications of the cells. Therefore, studies employing chemically defined xeno-free (XF) media for MSC cultures are extremely valuable. Many beneficial effects of MSCs have been attributed to their ability to regulate inflammation and immunity, mainly through secretion of immunomodulatory factors such as tumor necrosis factor-stimulated gene 6 (TSG6) and prostaglandin E2 (PGE2). However, MSCs require activation to produce these factors and since the effect of MSCs is often transient, great interest has emerged to discover ways of pre-activating the cells prior to their use, thus eliminating the lag time for activation in vivo. Here we present protocols to efficiently activate or prime MSCs in three-dimensional (3D) cultures under chemically defined XF conditions and to administer these pre-activated MSCs in vivo. Specifically, we first describe methods to generate spherical MSC micro-tissues or 'spheroids' in hanging drops using XF medium and demonstrate how the spheres and conditioned medium (CM) can be harvested for various applications. Second, we describe gene expression screens and in vitro functional assays to rapidly assess the level of MSC activation in spheroids, emphasizing the anti-inflammatory and anti-cancer potential of the cells. Third, we describe a novel method to inject intact MSC spheroids into the mouse peritoneal cavity for in vivo efficacy testing. Overall, the protocols herein overcome major challenges of obtaining pre-activated MSCs under chemically defined XF conditions and provide a flexible system to administer MSC spheroids for therapies.

Introduction

Mesenchymal stem/stromal cells (MSCs) have shown great potential for various regenerative medicine approaches. MSCs were initially isolated as a stromal component of bone marrow but have since been obtained from numerous other adult tissues, including adipose tissue^1,2,3. Interestingly, the main isolation method embraces the remarkable property of MSCs to adhere tightly onto tissue culture plastic in the presence of fetal bovine serum (FBS). Whilst this traditional isolation technique permits easy and rapid expansion of MSCs in two-dimensional (2D) culture, it is also very artificial and disregards significance of the native three-dimensional (3D) environment leading to potential loss of important cellular characteristics^4,5,6. Therefore, the study of MSCs in 3D cultures, which are more physiological than traditional 2D cultures, has emerged in search for "lost/diminished" MSC characteristics. Furthermore, great interest has risen to identify xeno-free (XF) chemically defined conditions for MSC culture and activation, and thus make the cells more amenable for clinical applications.

Many studies have been published demonstrating the 3D culture of MSCs both in biomaterials and as spherical aggregates or spheroids. MSCs in biomaterials were initially designed for tissue engineering approaches to replace damaged tissues with cell-seeded scaffolds, whereas spheroid cultures of MSCs were seen as a way to understand MSC behavior in vivo after administration of the cells for therapies in pre-clinical or clinical trials^4,5,7. Interestingly, MSCs form spheroids spontaneously when adherence to tissue culture plastic is not permitted^8,9,10. Traditionally, cell aggregation was facilitated by spinner flask methods or liquid overlay techniques, methods used initially in cancer biology in efforts to try to mimic the tumor microenvironment. More recently, additional methods have surfaced that demonstrate cell aggregation in culture dishes pre-coated with specific chemicals to prevent cell-to-plastic adhesion^4,5,6. One of the simplest and most economical methods to generate MSC spheroids is to culture them in hanging drops, a technique that was often used to produce embryoid bodies from embryonic stem cells. With hanging drop culture technique, cell adherence to the tissue culture plastic is prevented by suspending the cells in a drop of medium on the underside of a tissue culture dish lid and allowing gravity to facilitate cell aggregation in the apex of the drop. The spheroid size can be readily manipulated by changing the cell concentration or the drop volume, making hanging drop cultures particularly easy to control.

Early studies on the 3D culture of MSCs demonstrated radical differences in the characteristics of the cells in 3D compared to their 2D counterparts^6,8,9. At the same time, reports demonstrated that the beneficial effects of MSCs in vivo relied on their ability to become activated by micro-environmental cues and, in response, to produce anti-inflammatory and immunomodulatory factors¹¹. Interestingly, many of these factors such as prostaglandin E2 (PGE2), tumor necrosis factor-stimulated gene 6 (TSG6), and hepatocyte growth factor (HGF) were produced in much larger quantities by MSC spheroids than traditional 2D MSCs paving the way for the idea of using 3D cultures to activate the cells^8,12,13. Moreover, gene activation in 3D cultures appeared to recapitulate mechanisms, at least in part, of cell activation after injection into mice¹². By activating MSCs prior to their use in experiments, effects of the cells could be prolonged and more prominent as the traditional MSC effect in vivo is often delayed and transient, and can be described as "hit and run". During the past several years, important functional studies using MSC spheroids have demonstrated that they can suppress inflammatory responses and modulate immunity in vivo by influencing effector cells such as macrophages, dendritic cells, neutrophils, and T cells making spheroids an attractive form of primed MSCs^2,3. In addition, production of anti-cancer molecules, such as interleukin-24 (IL-24) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), are increased in 3D cultures of MSCs relative to monolayer MSCs, a phenomenon that could be exploited for targeted cancer therapies^8,10,14.

As the traditional MSC culture required not only the use of tissue culture plastic but also FBS, another hurdle to make MSC spheroids more amenable for clinical use had to be overcome. To tackle this hurdle, we recently showed formation of MSC spheroids under specific chemically defined XF conditions and established that the resulting MSC spheroids were activated to produce the same anti-inflammatory and anti-cancer molecules as the spheroids generated in conditions with FBS¹⁴. Here, these findings are presented in several detailed protocols that demonstrate the generation of pre-activated MSCs in 3D cultures using XF media. In addition, protocols are presented that describe effective ways to assess the activation levels of the MSCs in regards to their anti-inflammatory, immunomodulatory and anti-cancer effects, together with a practical method to deliver the intact spheroids into mice.

Protocol

1. MSC Isolation and Expansion

1. Obtain early passage MSCs from Center for the Preparation and Distribution of Adult Stem Cells (http://medicine.tamhsc.edu/irm/msc-distribution.html)¹⁵ as frozen vials. Alternatively, isolate MSCs from bone marrow aspirates following a routine protocol¹⁴ and store as frozen vials.
2. Prepare complete culture medium (CCM), which is minimal essential medium alpha (αMEM) supplemented with 15-20% premium select FBS, 2 mM L-glutamine, and 1x penicillin-streptomycin. Sterilize the medium by filtration.
3. Place 30 ml of CCM into a 150 mm cell culture dish and incubate the dish for 30 min at 37 °C in a humidified incubator containing 5% CO₂.
4. Incubate the frozen vial containing approximately 10⁶ MSCs for 2 min in a 37 °C water bath.
5. Transfer the contents of the vial to the culture dish using a 5 ml serological pipet and wash the vial several times with 1-2 ml CCM from the dish to capture residual cells. Place the dish overnight in an appropriate incubator.
6. Carefully wash the culture twice with room temperature phosphate buffered saline (PBS) and detach the MSCs with 3 ml of pre-warmed 0.25% trypsin/ethylenediaminetetraacetic acid (EDTA) solution. Detachment usually takes approximately 3-4 min in the incubator.
7. Neutralize the trypsin in the dish with 6 ml CCM pre-warmed to 37 °C and transfer the cell suspension into a 50 ml conical tube. Combine with washes of PBS to maximize cell yield.
8. Centrifuge the cells (450 x g) for 10 min at room temperature and aspirate the supernatant.
9. Re-suspend the cells in a small volume of CCM and count the viable cells using a hemocytometer and trypan blue.
10. Seed an appropriate number of 150 mm dishes at 100 cells/cm² in 30 ml of warm CCM and incubate the cultures until approximately 70-80% confluent, usually 7 days with medium changes every 2-3 days and 24-48 hr prior to harvest.
11. Harvest the cells as above with trypsin/EDTA and combine all the cells into a single conical tube with the appropriate medium. Centrifuge again and re-suspend the cells into a small volume of the same medium for cell counts. Use standard CCM (containing FBS) or various commercially available XF media formulations, here referred to as XF medium-1 (XFM-1) and XF medium-2 (XFM-2), both with and without human serum albumin (HSA, 13 mg/ml) supplementation.

2. Preparation of Activated MSC Spheroids in 3-D Hanging Drop Cultures under XF Conditions

1. To generate spheroids of approximately 25,000 cells, dilute the harvested MSCs into CCM, XFM-1, XFM-1 + HSA (13 mg/ml), XFM-2, or XFM-2 + HSA (13 mg/ ml) at 714 cells/µl.
2. Pipette 35 µl/drops of the cells onto the underside of a 150 mm inverted culture dish lid. Multiple drops can be easily pipetted by using a multichannel pipette.
   NOTE: If larger or smaller spheroids are desired, change the cell concentration or the drop volume (25-45 µl) appropriately.
3. Transfer 20 ml of room temperature PBS into the base of the dish and in one continuous and steady motion flip the lid, containing the drops, so that the drop apices face downward. Place the lid carefully on the base of the culture dish.
4. Transfer the dish into the incubator for 3 days without disturbing it to permit appropriate sphere assembly.
5. After 72 hr, begin the spheroid harvest by removing the lid carefully and inverting it so that the drops, once again, face upward.
6. Angle the lid to approximately 10-20° and push the drops, containing the spheroids, to the plate edge using a cell lifter.
7. Transfer the spheres and medium into a 15 ml conical tube with a 1,000 µl pipette. Use room temperature PBS and the pipette with the same tip to wash the plate and ensure maximal recovery of spheroids.
8. For real-time PCR assays (protocol 3), centrifuge the sphere suspension at 450 x g for 5 min at room temperature and aspirate the supernatant. Wash the spheroids with PBS and repeat both the centrifugation and aspiration steps. For delivery into animals (protocol 8), allow spheres to settle in the bottom of the tube (~3-4 min) without centrifugation.

3. Real-time PCR of Anti-inflammatory and Anti-cancer Markers

1. Use the RNA extraction kit with homogenizer columns and RNase-Free DNase Set to isolate DNA-free total RNA from monolayer MSCs (Protocol 1) and MSC spheroids (Protocol 2) that were washed with PBS and centrifuged.
2. Lyse at least 100,000 monolayer MSCs and 20 spheroids from each condition in separate 15 ml conical tubes with RLT buffer containing β-mercaptoethanol (1:100).
3. Transfer the thoroughly mixed lysates into 1.5 ml RNase-free tubes and place into a freezer (-80 °C) for at least 3 hr to aid in spheroid lysis.
4. Thaw the cell lysates on ice, vortex briefly, and follow the manufacturer's instructions for RNA isolation using a commercially available mammalian total RNA isolation kit.
5. Quantify and assess the quality of the isolated RNA using a spectrophotometer.
6. Use the High Capacity cDNA Reverse Transcription Kit or equivalent according to manufacturer's instructions to generate cDNA for real-time PCR.
7. Place cDNA samples, commercially designed real-time PCR primers/probes (Gene Expression Assays), and PCR Master Mix on ice. Use primers/probes for GAPDH (control), PTGS2 (COX-2, anti-inflammatory), TNFAIP6 (TSG6, anti-inflammatory), TRAIL (anti-cancer), and IL-24 (anti-cancer).
8. Prepare the real-time PCR reactions according to manufacturer's instructions for Gene Expression Assays and Fast Universal Master Mix.
9. Follow the guidelines for the real-time PCR instrument for generating the experiment documents and performing the run.
10. Analyze the data using the ΔΔC_T method by calculating the relative changes (relative quantity, RQ) in gene expression using GAPDH as the endogenous control and monolayer MSCs as a baseline^8,14.

4. Collection of CM for Assays of Immunomodulatory and Anti-cancer Potential

1. Harvest the spheroids and CM as described above into a 15 ml conical tube, using a cell lifter and a pipette, however, do not wash the dish lids with PBS as this will dilute the CM.
2. Centrifuge at 450 x g for 5 min at room temperature, carefully collect the cell-free supernatant with a pipette, and transfer the supernatant into 1.5 ml tubes.
3. Centrifuge at 10,000 x g for 5-10 min at room temperature, carefully collect the clarified CM with a pipette, and transfer the CM into new 1.5 ml tubes for storage in a freezer (-80 °C). Prepare aliquots of the CM prior to freezing when using it for multiple assays.
   NOTE: Prior to performing downstream assays, PGE2 concentration, a good indicator of anti-inflammatory potential of CM, can be determined using a commercial ELISA kit according to manufacturer's instructions. TSG6 concentration can be determined using a published protocol¹⁴.

5. Macrophage Assay to Assess the Anti-inflammatory Potential of MSC-CM

1. Prepare macrophage medium, which consists of Dulbecco's modified Eagle medium (DMEM) containing L-alanyl-L-glutamine and supplemented with 10% premium select FBS and 1x penicillin-streptomycin. Filter-sterilize the medium.
2. Obtain a frozen vial of approximately 10⁶ J774 mouse macrophages and incubate the vial for 2 min in a 37 °C water bath.
3. Transfer the contents of the vial into a 15 ml conical tube, add 9 ml of macrophage medium, and centrifuge for 5 min at 200-300 x g and at room temperature.
4. Aspirate the supernatant, suspend the macrophages into 1 ml of macrophage medium, and transfer the macrophages into a 150 mm petri dish containing 30 ml of macrophage medium.
5. Change the medium every 2 days and incubate the cells in the incubator until approximately 70-80% confluent.
   NOTE: The macrophages do not adhere tightly to the petri dish, thus care should be taken not to lose cells with medium change.
6. Harvest the macrophages by spraying the macrophage medium onto the cells with a pipette. Transfer the cells into a 50 ml conical tube and centrifuge for 5 min at 200-300 x g and room temperature.
7. Aspirate the supernatant and suspend the cells in a small volume of macrophage medium for cell counts with a hemocytometer. Adjust the concentration to 200,000 cells/ml with macrophage medium.
8. To start the set up for the macrophage assay, add 480 µl of macrophage medium into wells of a 12-well plate.
9. Add 20 µl of macrophage medium into non-stimulated control wells and 20 µl of non-conditioned or MSC-conditioned medium, prepared above, in experimental wells. Mix the medium in the wells by tapping the plate.
10. Transfer 500 µl of the macrophage cell suspension to the non-stimulated macrophage control wells.
11. Stimulate the remaining macrophages with addition of 1:500-1:1,000 of 0.1 mg/ml lipopolysaccharide (LPS) and incubate 5-10 min at room temperature.
12. Transfer 500 µl of the stimulated macrophages into the wells with non-conditioned or MSC-conditioned medium. Mix the wells by steadily rocking the plate several times.
13. Transfer the plate to an incubator for 16-18 hr.
14. Harvest the macrophage-conditioned medium and centrifuge at 500 x g for 5 min at room temperature.
15. Transfer the supernatant into new tubes and assay for tumor necrosis factor-alpha (TNF-α) and interleukin-10 (IL-10) content by commercial ELISA kits following the manufacturer's instructions.

6. Splenocyte Assay to Assess the Immunomodulatory Potential of Spheroid-CM

1. Prepare complete splenocyte medium, which consists of Rosewell Park Memorial Institute (RPMI)-1640 medium supplemented with 10% heat-inactivated FBS, 1x penicillin-streptomycin, and 2-mercaptoethanol. Filter-sterilize the medium.
2. Harvest spleens from euthanized-male BALB/c mice through an incision prepared on the left side of the abdomen over the spleen, approximately mid-way between the front and hind legs¹⁴. Transfer the spleen to a 70 µm cell strainer to isolate the splenocytes¹⁴.
3. Isolate the splenocytes/lymphocytes by pushing the spleens through the 70 µm strainer into a sterile petri dish using the soft rubber/plastic end of a plunger from a 2-3 ml syringe¹⁴. Use a grinding circular motion to dissociate the spleens.
4. Wash the cell strainer several times with cold PBS and transfer cells from the petri dish into a 50 ml conical tube. Fill the tube with cold PBS and centrifuge (500 x g) at 4 °C for 10 min.
5. Aspirate the supernatant and clear the cells from erythrocytes by incubation with 5-10 ml red blood cell lysis solution (per spleen) for 5-10 min.
6. Stop the lysis by adding cold PBS to the tube and centrifuge for 5-10 min at 500 x g and at 4°C.
7. Suspend the isolated cells in complete splenocyte medium, filter through a 40 µm strainer, and count the cells using a hemocytometer. Add splenocyte medium to the cell suspension to achieve a final cell concentration of 10⁶ cells/ml
   NOTE: This technique typically yields approximately 3 x 10⁷ splenocytes per mouse spleen.
8. Stimulate the appropriate number of splenocytes with 2 µg/ml anti-CD3e antibody for 5 min.
   NOTE: The quantity of splenocytes required depends on the number of experimental conditions as determined by the investigator (see step 6.9). For every experimental sample/well, 10⁶ splenocytes are required.
9. Transfer 10⁶ splenocytes (stimulated or non-stimulated) into wells of a 12-well plate and add non-conditioned (controls) or MSC-CM into wells at final dilution of 1:10-1:20.
10. Harvest the splenocyte CM after 24 hr and centrifuge at 500 x g for 5 min at room temperature.
11. Transfer the supernatant into new tubes and assay for interferon gamma (IFN-γ) content by commercial ELISA kit following the manufacturer's instructions.

7. Prostate Cancer Assay to Assess the Anti-cancer Potential of Spheroid-CM

1. Prepare complete RPMI growth medium that is RPMI-1640 medium supplemented with 10% FBS, 1x penicillin-streptomycin.
2. Obtain a frozen vial of LNCaP prostate cancer cells and incubate the vial for 2 min in a 37 °C water bath.
3. Transfer the contents of the vial into a culture dish containing 30 ml of complete RPMI growth medium using a motorized pipettor and a 5 ml serological pipet. Wash the vial several times with the medium from the dish and place the dish into the incubator.
4. Just before the cells reach confluency, wash the culture twice with room temperature PBS and detach the LNCaP cells with 3 ml of pre-warmed 0.25% trypsin/EDTA solution.
5. Neutralize the trypsin in the dish with the complete RPMI growth medium and transfer the cells together with washes into a 50 ml conical tube.
6. Centrifuge the cells at 450 x g for 10 min at room temperature and aspirate the supernatant.
7. Re-suspend the cells into small volume of the complete RPMI growth medium and count the cancer cells using a hemocytometer.
8. Transfer LNCaP cells into a 96-well dish at 5,000 cells/well in 120 µl of complete RPMI growth medium containing 25% of non-conditioned or spheroid-CM. Incubate in the incubator for 72 hr.
9. To perform a cell growth assay using a cell proliferation assay kit, aspirate the medium from wells and transfer the plate into a freezer (-80 °C) for at least 3 hr.
   1. Thaw the plate and lyse the cells in each well by adding 100 µl of cell lysis reagent containing 180 mM NaCl, 1 mM EDTA, and 0.2 mg/ml RNase A.
   2. For a standard curve, lyse known amounts of LNCaP cells as described above.
   3. After 1 hr, add 100 µl of 1x cell lysis buffer containing 1:100 dilution of cell proliferation assay reagent and measure the fluorescence in each well to determine the cell amount.

8. Intraperitoneal Delivery of Intact Spheroids

1. Prepare spheroid MSCs as described above (protocol 2) and resuspend the spheres in sterile Hank's Balanced Salt Solution (HBSS) supplemented with 0.2-0.5 % HSA to maintain XF conditions. HSA in the solution helps to prevent sphere adhesion to plastic during injection.
2. Transfer 40-120 spheroids into 15 ml conical tubes and wash the cells by adding 6 ml HBSS/HSA to the tubes. Allow 3-4 min for spheres to descend. Prepare an independent conical tube of spheroids for each animal. Also, prepare additional tubes of spheroids for assays determining spheroid retention (see 8.18 below) and production of therapeutic factors after transfer (see 8.19 below) if desired.
3. Aspirate the supernatant, overlay the spheroids with 100-200 µl of sterile HBSS supplemented with 0.2-0.5% HSA, and place the tubes on ice.
4. Briefly anesthetize the animals with 2% isoflurane in 100% oxygen until the disappearance of pinch-toe reflex for approximately 2 min in the induction chamber.
5. Place the animal inside the BSL-2 cabinet, dorsal aspect down (ventral side facing up), with the continuous flow of 1.5% isoflurane/oxygen mixture via a nose cone.
6. Clean the lower mouse abdomen with an antiseptic such as 70% alcohol.
7. Remove the cap of the 20 G intravenous (iv) catheter and perform the intraperitoneal injection with the catheter-stiletto assembly. Use one hand to hold the mouse skin and another to perform the injection. Locate the point of injection approximately in the middle of an artificial line connecting a flexed knee joint and genital area with the needle tip pointing toward the midline.
   NOTE: The catheter-stiletto assembly has a higher resistance than a regular needle, therefore care has to be taken as not to inject the needle too deep into the abdomen, which could result in injury to internal organs. Penetration of the skin and then muscles/peritoneal membrane can be felt during catheter placement.
8. Gently remove the metal stiletto/needle from the catheter-stiletto assembly. Check the stiletto for blood, urine and feces and discard it. Blood on the needle indicates puncture of internal organ(s).
9. Hold the plastic catheter in an upright position during the injections to permit fluid flow.
10. Confirm the proper placement of the plastic catheter by injecting approximately 100 µl of sterile HBSS/HSA through the catheter using a pipette with a 100-200 µl tip. Place the end of the pipette tip inside the catheter syringe adapter forming a tight seal. Slowly inject the fluid inside the peritoneal cavity.
    NOTE: The fluid in a properly positioned catheter will freely flow inside the peritoneal cavity with only minor adjustment of the catheter. Improper placement of the catheter (subcutaneous or intra-intestinal, or if the tip of the needle injured peritoneal organs such as kidneys) will increase resistance and reduce flow. Subcutaneous placement will result in a formation of an obvious "bubble" under the skin.
11. Collect the MSC spheroids, prepared in 100-200 µl of HBSS/HSA (step 8.3), using a 200 µl pipette tip, and transfer the entire volume of spheres (40-120 spheres) into the peritoneal cavity through the catheter as described above.
12. Wash the tube containing spheres with 100 µl of HBSS/HSA using the same tip as above to collect any residual spheroids and inject them into the peritoneal cavity.
13. (Optional) Inject an additional 100 µl of HBSS/HSA into the peritoneal cavity to wash the catheter.
14. Place the gauze pad soaked in antiseptic over the catheter and slowly remove the catheter from the peritoneal cavity and discard it.
15. Gently massage the peritoneal cavity with the fingers to ensure even distribution of the spheroids.
16. Let the animal recover under close supervision and watch for bleeding from the injection site.
17. Inspect the catheter and 15 ml tube for any remaining spheroids. It is normal to observe a few spheres in the catheter/tube.
    NOTE: The technique described for MSC spheroid delivery can be adopted for use in normal animals or in a variety of injury models. This technique has been successfully used to inject spheroids in mouse corneal injury and endotoxemia models, as well as in a zymosan-induced peritonitis model⁸.
18. To quantify the number of retained spheroids (optional), use a DNA-based cell number quantification kit/reagent. Hold the used catheter over the 15 ml tube and vigorously dispense HBSS/HSA through the catheter to force any remaining spheres back into the 15 ml tube.
    1. Centrifuge the 15 ml tube at 500 x g for 5 min at room temperature and aspirate the supernatant. Transfer the tube containing the spheroid pellet into a freezer (-80 °C) for at least 3 hr.
    2. Thaw the tube and lyse the spheres by adding 500 µl of cell lysis reagent containing 180 mM NaCl, 1 mM EDTA, and 0.2 mg/ml RNase A.
    3. For a control, freeze and lyse a known amount of spheroids (preferably equivalent to the number of spheroids prepared for a single injection) as described above.
    4. After 1 hr, transfer 100 µl of the cell lysate, in duplicate, into a 96-well microplate and add to each well 100 µl of 1x cell lysis buffer containing a 1:50 dilution of cell proliferation assay reagent from the kit. After 10 min, measure the fluorescence in each well to determine the number of spheres that were not injected by comparing the experimental sample(s) with the control sample.
19. Evaluate the activation level of spheroids prepared for injection (optional) by measuring concentration of PGE2 and TSG6 produced by transferred spheres. Transfer 40-120 spheres through the catheter, as described above, into a 12-well or 6-well plate containing 1 or 2 ml αMEM supplemented with 2% FBS and 1x penicillin/streptomycin. Place the plates in the incubator for 6 hr.
    1. Transfer the medium from the wells after 6 hr into 1.5 or 15 ml tubes and centrifuge the tubes at 450 x g for 5 min at room temperature.
    2. Transfer the cell-free supernatant into fresh 1.5 ml tubes using a pipette and centrifuge at 10,000 x g for 5-10 min at room temperature.
    3. Transfer the clarified CM (supernatant) into new 1.5 ml tubes and assay for PGE2 concentration (good indicator of anti-inflammatory potential of CM) using a commercial ELISA kit according to manufacturer's instructions. TSG6 concentration can be determined using a published protocol¹⁴.

Results

In the current work, hanging drop cultures were employed to generate compact spherical micro-tissues or 'spheroids' of activated MSCs under XF conditions. The investigational roadmap in Figure 1 depicts that MSCs are encouraged to self-assemble into spheroids when suspended in hanging drops for 72 hr, after which the spheroids, or the CM loaded with sphere-derived therapeutic factors, can be collected and potentially utilized in both research and clinical applicat...

Discussion

The optimal MSC for use in some research and clinical applications should be highly activated to maximize their benefit, and preferentially prepared under chemically defined XF conditions to minimize the delivery of potential antigens from xenogeneic medium components such as FBS. In the protocols described here, we have shown methods to 1) activate MSCs in 3D culture by formation of spheroids, 2) achieve the 3D activation of MSCs under XF conditions, 3) evaluate the activation levels of spheroid MSCs in regards to their...

Materials

Name	Company	Catalog Number	Comments
MEM-α (minimal essential medium alpha)	ThermoFisher/Gibco	12561049; 12561056; 12561072	minimal essential medium for preparation of MSC growth medium (CCM)
FBS (fetal bovine serum), premium select	Atlanta Biologicals	S11595; S11510; S11550; S11595-24	component of complete culture media for all types of cells
L-glutamine	ThermoFisher/Gibco	25030081; 25030149; 25030164	component of complete culture media for all types of cells
Penicillin/Streptomycin	ThermoFisher/Gibco	15070063	component of complete culture media for all types of cells
Sterilization Filter Units, 0.22 µm PES membrane	MilliporeSigma	SCGPU01RE; SCGPU02RE; SCGPU05RE; SCGPU10RE; SCGPU11RE	media sterilization
150 mm cell culture dish	Nunc	D8554 SIGMA	cell culture
Thermo Forma water-jacketed CO2 humidified incubator	Thermo Fisher	Model 3110	incubation of cultured cells
Early passage MCSs	Center for the preparation and Distribution of Adult Stem Cells at The Texas A&M Health Science Center College of Medicine Institute for Regenerative Medicine at Scott & White	NA	preparation of 2D and 3D cultures of MSCs
water bath	VWR	89501-468	warming media to 37 °C
Pipettes	Eppendorf	492000904	manual liquid handling
Pipete-Aid	Drummond Scientific Company	4-000-300	handling sereological pipetes
Costar sterile serological pipet (5, 10, 25 and 50 ml)	Corning	4487; 4101; 4251; 4490	liquid handling
PBS (phosphate buffered saline), pH 7.4	ThermoFisher/Gibco	10010023; 10010072; 10010031; 10010049	cell culture processing
0.25% trypsin/EDTA solution	ThermoFisher/Gibco	25200056; 25200072; 25200114	lifting adherent cells and dispersing cell aggregates
15 ml conical tube	Corning/BD Falcon	352097	cell centrifugation
50 ml conical tube	Corning/BD Falcon	352098	cell centrifugation
Eppendor refrigerated centrifuge	Eppendorf/Fisher Scientific	Model 5810R	cell centrifugation
hemocytometer	Fisher Scientific	26716	cell counting
trypan blue	Sigma-Aldrich	T8154 SIGMA	dead cell exclusion during cell counting in hemacytometer
Defined xenofree MSC medium-1 (XFM-1)	ThermoFisher/Gibco	A1067501	Xeno-free media specifically formulated for the growth and expansion of human mesenchymal stem cells
Defined xenofree MSC medium-2 (XFM-2)	Stem Cell Technologies	5420	Defined, xeno-free medium for human mesenchymal stem cells
HSA (Human serum albumin)	Gemini	800-120	Component of xeno-free MSC media
rHSA (recombinant Human serum albumin)	Sigma-Aldrich	A9731 SIGMA	Component of xeno-free MSC media
Total RNA isolation Mini Kit	Qiagen	74104	Total RNA extraction
Qiashredder	Qiagen	79654	Sample homogenization prior to total RNA extraction
RNAse-free DNase Set	Qiagen	79254	On-column DNA elimination during total RNA extraction
β-mercaptoethanol	Sigma-Aldrich	M6250 ALDRICH	inhibition of RNAses in RLT buffer
Vortex	VWR	97043-562	mixing sample
Spectrophotometer	Biorad	NA	RNA concentration and quality
High capacity cDNA Reverse Transcription Kit	ThermoFisher/Applied Biosystems	4368814	transcription of total RNA into cDNA
Gene Expression Assays	ThermoFisher/Applied Biosystems	varies	primer/probe combination for real-time PCR
Fast Universal PCR Master Mix	ThermoFisher/Applied Biosystems	4352042; 4364103; 4366073; 4367846	master mix for real-time PCR reaction
Real-time PCR system (ABI Prism 7900 HT Sequence Detection System)	ABI Prizm	NA	real-time PCR
1.5 ml centrifuge tube	Eppendorf	22364111	cell centrifugation, sample collection and storage
(-80 °C) freezer	Thermo Fisher	Model Thermo Forma 8695	sample storage
PGE2 (Prostaglandin E2) ELISA Kit	R&D Systems	KGE004B	estimation of cytokine concentration in the sample
DMEM (Dulbecco’s modified Eagle medium)	ThermoFisher/Gibco	10566-016; 10566-024;10566-032	macrophage culture media
J774 mouse macrophages	ATCC	TIB-67	mouse macrophage cell line
12-well plate	Corning	3513	in vitro macrophage stimulation
LPS (lipopolysaccharide)	Sigma-aldrich	L4130	in vitro macrophage stimulation
Mouse TNF-a ELISA kit	R&D Systems	MTA00B	estimation of cytokine concentration in the sample
Mouse IL-10 (interleukin 10) ELISA kit	R&D Systems	M1000B	estimation of cytokine concentration in the sample
RPMI-1640 medium	ThermoFisher/Gibco	11875-085	splenocyte culture media
BALB/c mice	The Jackson Laboratory	651	in vivo spheroid delivery; splenocyte preparation
Anti-Mouse CD3e Functional Grade Purified	eBioscience	145-2C11	in vitro splenocyte stimulation
70 μm strainer	Corning	352350	Splenocyte preparation
Red blood cell lysis solution (1x)	Affymetrix eBioscience	00-4333	removal of red blood cells during splenocyte isolation
Mouse IFN-γ (interferon gamma) ELISA kit	R&D Systems	MIF 00	estimation of cytokine concentration in the sample
LNCaP prostate cancer cells	ATCC	CRL-1740	study the effect of 3D MSCs on cancer cell lines in vitro
DNA-based cell proliferation assay kit	ThermoFisher	C7026	cell number measurement based on DNA content
NaCl	Sigma-Aldrich	S5150	component of lysis reagent
EDTA (ethylenediaminetetraacetic acid)	ThermoFisher	FERR1021	calcium chelator, component of lysis reagent
Rnase A	Qiagen	19101	RNA degradation for measurement of DNA
Filter-based multi-mode microplate reader	BMG Technology	NA	Microplate assays (ELISA, cell quantification, etc.)
HBSS (Hanks balanced salt solution), no calcium, no magnesium, no phenol red	ThermoFisher/Gibco	14175079	resupsension of MSC spheroids prior to in vivo injections
Isoflurane	MWI Vet Supply	502017	Anesthesia for in vivo injections
Oxygen, compressed gas	Praxair	NA	For use with isoflurane
Thermo Forma BSL-2 cabinet	Thermo Fisher	Model 1385	Sterile cell culture
Safety I.V. catheter/needle stiletto, 20 G , 1 inch	Terumo	SR*FNP2025	Delivery of shperoids into peritoneal cavity
Sterile micropipette tips	Eppendorf	varies	liquid/cells handling