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.
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.
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 tissue1,2,3. 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 characteristics4,5,6. 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 trials4,5,7. Interestingly, MSCs form spheroids spontaneously when adherence to tissue culture plastic is not permitted8,9,10. 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 adhesion4,5,6. 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 counterparts6,8,9. 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 cells8,12,13. 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 MSCs2,3. 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 therapies8,10,14.
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.
1. MSC Isolation and Expansion
2. Preparation of Activated MSC Spheroids in 3-D Hanging Drop Cultures under XF Conditions
3. Real-time PCR of Anti-inflammatory and Anti-cancer Markers
4. Collection of CM for Assays of Immunomodulatory and Anti-cancer Potential
5. Macrophage Assay to Assess the Anti-inflammatory Potential of MSC-CM
6. Splenocyte Assay to Assess the Immunomodulatory Potential of Spheroid-CM
7. Prostate Cancer Assay to Assess the Anti-cancer Potential of Spheroid-CM
8. Intraperitoneal Delivery of Intact Spheroids
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 applications. The large number of cells needed for sphere production can be acquired within one week by seeding the MSCs at low density, typically 100-200 cells per cm2, and then expanding the cells for 6-7 days until approximately 80% confluent (Figure 2). Cell growth kinetics, determined by counting viable cells daily, indicated that a robust expansion phase (day 3-7) follows a brief lag phase of 1-2 days (Figure 2C). MSCs in passage 2 or 3 typically yield 50-100 million cells from a single vial of 1 million cryopreserved cells when cultured in CCM.
Following expansion and harvest, the MSCs are suspended at high cell concentration to generate spheroids, typically 500-1,000 cells per µl. Hanging drop cultures are prepared by transferring 25-40 µl droplets of medium containing the MSCs onto the underside of a tissue-culture dish lid, which is subsequently flipped and appropriately positioned onto the base of the dish (Figure 3). When suspended in 35 µl drops of CCM at 714 cells per µl (i.e. 25,000 cells per drop), MSCs first assemble into small aggregates that eventually coalesce to generate a single compact sphere at 72 hr in the apex of the drop (Figure 3C). Spheroids also form over 72 hr in several types of commercially available XF media optimized for MSC expansion, here referred to as XFM-1 and XFM-2. However, formation of single compact spheres in XFM-1 requires supplementation with 13 mg/ml human serum albumin (HSA), a concentration that reflects the estimated total protein content in CCM (Figure 3D). Single compact spheres do not readily form in basic XFM-1 without HSA or in protein-free αMEM (Figure 3D). During sphere assembly, MSC phenotype changes radically (Figure 4), and when in the appropriate chemically-defined XF medium (i.e. XFM-1 + HSA), expression of numerous anti-inflammatory factors are upregulated including PGE2 and TSG6 (Figure 4C). However, production of TSG6 and PGE2 is not markedly augmented in MSCs cultured as spheres in XFM-2 or XFM-1 without HSA (Figure 4C). Notably these anti-inflammatory factors, as well as the anti-cancer factors TRAIL and IL-24, were highly upregulated in spheroid MSCs relative to monolayer MSCs, when the spheres were cultured in XFM-1 supplemented with either recombinant HSA (rHSA) or clinical grade HSA (cHSA) prepared from human blood (Figure 4D).
To evaluate the therapeutic potential of MSC spheres prepared in various media formulations, several practical tests are performed (Figure 5). The immune-modulatory properties of spheroids are first determined in vitro by measuring the effects of sphere CM on levels of cytokines produced by LPS-stimulated macrophages and CD3-stimulated splenocytes/lymphocytes (Figure 5). CM from MSC spheres cultured in CCM and XFM-1/HSA markedly suppressed production of macrophage TNFα and splenocyte IFNγ, while at the same time enhanced levels of the anti-inflammatory cytokine IL-10 (Figure 5A and 5B). The anti-cancer properties of spheres are evaluated by measuring the effects of sphere CM on growth and morphology of LNCaP prostate cancer cells. The XF medium XFM-1/HSA effectively reduced LNCaP proliferation similar to FBS-containing CCM (Figure 5C and 5D).
Importantly, intact MSC spheres can be administered into the peritoneal cavity of mice to test the anti-inflammatory activity of the cells in vivo (Figure 6). For these experiments, spheres are prepared for injection in HBSS containing a low concentration of HSA, which minimizes their adhesion to plastic tubing while preserving a XF state. Subsequently, spheres can be easily injected after transfer from the collection tube (Figure 6A), using a pipette (Figure 6B), through a needle/catheter assembly (Figure 6C and 6D) appropriately positioned for a standard intraperitoneal injection. Using this approach, MSC spheroids can be regularly transferred at an efficiency greater than 90% (Figure 6E and 6F) without disrupting their integrity (Figure 6G and 6H). Improper positioning of the catheter tip within the peritoneal cavity leads to poor sphere delivery and high retention (Figure 6F). Importantly, level of sphere retention within the pipette/catheter can be qualitatively determined by visual inspection, or quantitatively by collecting/lysing the retained spheres and measuring cell number with a commercially available DNA-based cell quantification reagent/kit (Figure 6F). Post-injection sphere retention analysis helps create inclusion/exclusion criteria during experimentation. Notably, the ability for CCM and XFM-1/HSA spheroids to secrete high levels of TSG6 and PGE2 are maintained at least 6 hr after transfer of the spheres from hanging drop cultures (Figure 6I). Similar to their in vitro effects, XFM-1/HSA spheres injected into the peritoneal cavity of mice, immediately following induction of systemic inflammation by iv administration of LPS, enhanced PGE2 and IL-10 levels in the peritoneum while decreasing pro-inflammatory TNFα (Figure 6J).
Figure 1: Schematic representation of the platform developed for harnessing the secretome of MSCs primed as spherical micro-tissues under XF conditions. Following expansion as monolayer cultures (1), the MSCs are suspended in hanging drops from the lid of a culture dish (2) at high cell concentration to activate/prime the cells. After 72 hr, both the (3) conditioned medium (CM) and (4) intact spheroids can be harvested from the droplets and utilized in various downstream applications. The CM is rich in immune-modulating factors, as well as other therapeutic components. The compact spheroids that form in hanging drops can be easily transferred using a pipette (5) and effectively administered in vivo through a catheter (6). Please click here to view a larger version of this figure.
Figure 2: Growth characteristics of bone marrow-derived MSCs in monolayer cultures. MSCs in passage 2 were seeded at low density (100 cells per cm2) in 15 cm dishes (15,000 cells per dish) and cultured for 7 days in CCM. Medium was changed on day 3 and then again on day 5 or 6 (24-48 hr prior to harvest). Representative phase-contrast micrographs of MSCs 3 days (A) and 7 days (B) after plating the cells. Scale bar = 200 µm. (C) Growth kinetics of MSCs obtained from 3 donors were determined by counting the number of viable adherent cells daily from day 2 to day 7. Please click here to view a larger version of this figure.
Figure 3: Preparation of hanging drop cultures for priming MSC spheroids in XF medium. (A) Photograph depicting the technique to rapidly layer hanging drops on the underside of a tissue culture dish lid. (B) Photo illustrating hanging drops after inversion and positioning of the lid onto the dish base. (C) Time-dependent changes in the aggregation of 25,000 MSCs in a hanging drop as visualized by phase-contrast microscopy. The objective was focused on the apex of the drop. Scale bar = 500 µm. (D) Images of MSC spheres formed after 3 days in hanging drop cultures using various media formulations including αMEM with and without FBS (i.e. CCM), XFM-1 with and without HSA, and XFM-2 with and without HSA. Scale bar = 200 µm. Data in panel D were obtained from the original article with permission from the publisher14. Please click here to view a larger version of this figure.
Figure 4: Evaluation of MSC activation in 3-D XF cultures. (A) Spheres/CM are harvested after 72 hr by inverting/tilting the dish lid and forcing the droplets to the edge using a cell lifter. Spheres assembled from 25,000 cells can be easily visualized during collection. (B) Photograph of approximately 500 spheres collected from lids of numerous tissue culture plates. Spheres rapidly descend to the bottom of tube without centrifugation. (C) Level of PGE2 secreted from MSC spheres after 72 hr was measured by ELISA. Real-time RT-PCR was used to measure TSG6 levels. Both TSG6 and PGE2 are valuable markers for screening MSC activation in spheroids. The XF medium XFM-1 + HSA showed high level of PGE2 and TSG6 production (red boxes). Data are shown as mean ± SD and were analyzed by Student's t-test (*** p < 0.001, compared to αMEM sample). (D) PGE2 ELISA and real-time RT-PCR assays on spheres produced in CCM and XFM-1 supplemented specifically with 13 mg/ml recombinant HSA (rHSA), or clinical grade HSA (cHSA) prepared from human venous blood. Fold changes were determined from adherent monolayer MSCs (RQ = 1). Data are shown as mean ± SD and were analyzed by Student's t-test (** p < 0.01, *** p < 0.001, compared to monolayer MSCs). Some data in panels C and D were obtained from the original article with permission from the publisher14. Please click here to view a larger version of this figure.
Figure 5: In vitro functional assays for evaluating the anti-inflammatory and anti-cancer properties of MSC-CM collected from hanging drop cultures. (A) Representative results from ELISAs showing the effects of sphere CM (CCM and XFM-1/HSA) on production of TNFα and IL-10 by LPS-stimulated mouse macrophages (MQ). (B) Representative results from ELISA showing the effects of sphere CM (CCM and XFM-1/HSA) on production of IFNγ by mouse splenocytes (Spl) stimulated with an anti-CD3 antibody. (C) Phase contrast micrographs of LNCaP prostate cancer cells treated with basic CCM and XFM-1/HSA or conditioned medium (CM) from MSC spheres prepared in CCM and XFM-1/HSA. LNCaP cells were cultured in RPMI medium (control). Scale bar is 200 µm. (D) Quantitative changes in growth of the LNCaP prostate cancer cells was determined with a commercially available DNA-based cell quantification reagent/kit. Dotted line indicates seeding density of 5,000 cells per well. Data in all panels are shown as mean ± SD, and were analyzed by Student's t-test (*** p < 0.001). Some data in all panels were obtained from the original articles with permission from the publisher14. Please click here to view a larger version of this figure.
Figure 6: Efficient transfer of intact MSCs spheroids, prepared under XF conditions, using a 20G iv needle/catheter system. (A) Representative photograph of a 15 ml conical tube with XFM-1/HSA MSC spheres prepared for injection in HBSS containing 0.2% HSA. (B) Representative photograph of the MSC spheres after aspiration into a 200 µl pipette tip. (C) Image of the 20 G needle/catheter assembly used to administer intact MSC spheroids into mice. (D) Image demonstrating proper positioning of the pipette tip into the adapter of the polyurethane catheter. (E) Image of the MSC spheres immediately after passing them through the catheter into a culture dish. Approximately 95 spheres out of 100 were transferred. (F) Efficiency of sphere delivery into the peritoneal cavity of mice was determined by collecting/lysing spheres retained in the pipette/catheter and measuring cell numbers, via a commercial DNA-based cell quantification assay, relative to the number of cells obtained from a full complement of lysed spheres. Dotted red line indicates a transfer efficiency of 90%. Poor sphere delivery (71%) was observed with one injection (arrow). (G) Phase-contrast micrographs of spheres in panel E (magnified view). Scale bar = 200 µm. (H) Phase-contrast micrograph of a XFM-1/HSA sphere 16 hr after transfer onto a tissue culture dish. The fibroblastic, spindle-shaped morphology of MSCs is maintained in cells that migrated out of the sphere. Scale bar = 200 µm. (I) ELISA assays for the anti-inflammatory factors TSG6 and PGE2 were performed on CM collected 6 hr after transfer of the spheres into 6-well plates containing 2 ml αMEM/2% FBS. Spheres prepared in the XF medium XFM-1/HSA showed highest level of TSG6 and PGE2 secretion (red boxes). Data, expressed as mean ± SD, were analyzed by Student's t-test (*** p < 0.001, compared to αMEM sample). Some data were obtained from the original article with permission from the publisher14. (J) Representative graphs illustrating the ability for spheroids, injected into the peritoneal cavity, to increase peritoneal PGE2 and IL-10 while decreasing level of TNFα in mice challenged by intravenous injection of endotoxin (LPS). Samples were obtained by peritoneal lavage 6 hr after induction of inflammation and delivery of 80 spheroids. Data, expressed as mean ± SD, were analyzed by Student's t-test (* p < 0.05, ** p < 0.01, compared to control). Please click here to view a larger version of this figure.
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 anti-inflammatory, immune-modulatory, and anti-cancer potential, and 4) deliver activated MSCs as intact spheroids into mice.
MSCs are multipotent stem cells that can be isolated via plastic adherence from numerous adult tissues including bone marrow and adipose tissue. MSCs are easily propagated on tissue culture plastic under relatively high (10-20%) FBS concentrations, express a distinct set of surface markers, and can be differentiated at least into adipogenic, osteogenic, and chondrogenic lineages1,16,17,18. MSCs have been demonstrated to exert beneficial effects in various animal models of human diseases, even though they appear to persist only transiently after their administration in vivo. The effect of MSCs has therefore been called "hit and run" and is often mediated by the anti-inflammatory and immunomodulatory paracrine factors, such as PGE2, TSG-6, and indoleamine 2,3-dioxygenase (IDO), secreted by MSCs2,3,11,19,20,21. However, in order to produce these factors, MSCs require activation, either through signals from a damaged tissue or from immune cells of the recipient. Subsequently, there has been an emerging interest to develop MSC pre-activation or priming protocols before delivery of the cells into animals or patients22. Also, the presence of antigenic FBS components in standard MSC preparations has raised concerns. Therefore, studies have been conducted to determine optimal chemically defined XF conditions for MSC cultures. In our recent work, we first showed that MSCs can be activated in 3D by formation of spheroids, and then discovered that cell activation can also be achieved under specific XF conditions8,12,13,14.
3D cell culture techniques have been employed for decades with the goal of providing genuine physiological conditions in cell-based research. Cultures in 2D disregard the 3D nature of tissues and therefore do not always recapitulate the cell-to-cell and cell-to-matrix interactions important in cell signaling. Many 3D culture techniques use rotating vessels, spinner flasks, or various non-adherent surfaces, however, the biggest drawback in many of these methods is the heterogeneity of the generated spheroid size and/or the requirement of specific expensive equipment. Research has also been performed using hanging drop cultures that essentially encourage MSCs to spontaneously aggregate into a single spheroid4,5,6,7,8,9,23. Notably, hanging drop cultures support assembly of relatively uniform micro-tissues/aggregates, with the added benefit of being able to easily manipulate size of the cell aggregate by adjusting cell concentration and/or drop volume. Moreover, mastery of the hanging drop culture technique does not require extensive training. Drops of 25-40 µl can be easily prepared with MSCs at high cell concentrations, typically 500-1,000 cells per µl. Drops smaller than 20 µl tend to evaporate quickly, and those larger than 45 µl often smear across the lid during inversion. However, when flipping a lid containing droplets of any size, speed and directionality are critical parameters to consider for maintaining surface tension and appropriate shape of the drop/spheroid. Uninterrupted culture and proper airflow in the incubator are also important for ensuring the MSCs aggregate into a single sphere within each drop.
A disadvantage of this technique is that plates containing hanging drops should not be stacked in the incubator or moved during sphere assembly, making scale-up for patient therapies difficult. Moreover, changing medium in hanging drop cultures is extremely challenging, thus long-term cultures should be avoided. In addition, the low media-to-cell ratio in hanging drops can cause nutrient depletion and waste accumulation eventually leading to the loss of important cellular functions and/or cell death.
However, with proper hanging drop technique, we have demonstrated that MSCs in spheroids become highly active or primed, in that the cells become factories of the powerful anti-inflammatory molecules PGE2 and TSG6, as well as the anti-cancer molecules IL-24 and TRAIL. We have shown that MSC spheroids are indeed anti-inflammatory and immunomodulatory, and have anti-cancer properties superior to their 2D monolayer counterparts in numerous functional assays using cultured macrophages, splenocytes, and prostate cancer cells8,12,13,14. Furthermore, we have shown that MSC spheroids can exert potent anti-inflammatory effects when administered into the peritoneal cavity of mice with peritonitis8. Specifically, we showed that large spheroids of approximately 400-500 µm in diameter (25,000-30,000 MSCs each) can be efficiently delivered in a small volume of HBSS using a 20G needle/catheter assembly and a standard pipette. With this technique, improper catheter placement, which results in high resistance to fluid flow, can be easily determined prior to spheroid transfer by first injecting a small volume of HBSS/HSA as described in the protocols. The spheroids in a properly positioned catheter will flow freely, provided that the HBSS is supplemented with HSA to minimize sphere adhesion to the plastic tubing, and can be easily visualized. Moreover, this technique prevents shear stress on cells which can occur using a standard needle/syringe for injection, and avoids the need for a surgical incision which carries a higher risk of infection and is more technically challenging. One major disadvantage is that large numbers of spheroids can only be injected into spacious body cavities, such as the peritoneal cavity, as the resistance against sphere transfer through the catheter is high in constricted areas.
We also recently demonstrated that the same level of MSC activation in spheroids could be achieved by using a specific commercially available XF medium, referred to here as XFM-1, as long as it was supplemented with HSA obtained from human blood or prepared through recombinant techniques14. It is important to note here that MSC spheroids do not produce high levels of PGE2 and TSG6 in all types of XF media14, probably because most XF media for MSCs were initially formulated for optimal expansion of the cells in 2D. It is also important to note that different types of HSA vary in their potency14. In addition, the absolute level of PGE2 detected in CM of activated spheroids can vary slightly as the ELISA employed for PGE2 is indeed a competition assay. Thus, it is crucial to include proper controls in every PGE2 ELISA and to avoid directly comparing data between samples assayed at different times or in different plates. Moreover, MSCs must be thoroughly washed in XF medium prior to preparing hanging drops in order to remove residual CCM and, therefore, limit carryover of FBS components. The protocols described here can be easily adopted to evaluate other media formulations on sphere formation and therapeutic gene expression.
Clearly, numerous cell-signaling events that do not normally occur in 2D MSCs are set in motion when MSCs are cultured in 3D. Cell-to-cell and cell-to-matrix interactions, mediated by cadherins and integrins guide spheroid formation and compaction24,25,26 and are likely critical for spheroid therapy. As the cells aggregate and compact into spheroids, various stress signals, including minor apoptosis, aid in the process of MSC activation that results in production of numerous potentially therapeutic factors4,5. We previously showed the critical role of autocrine IL-1 signaling in MSC activation and production of PGE2 and TSG612. While much remains unknown about the various signaling pathways that aid in MSC activation, the hanging drop culture platform affords a practical way to study this phenomenon and improve MSC-based therapies. Overall, we have described, in the protocols here, the means of activating or priming MSCs in 3D cultures, under chemically-defined XF conditions, to produce anti-inflammatory, immunomodulatory, and anti-cancer factors. We have also described how these intact MSC spheroids can be delivered in vivo.
The authors have nothing to disclose.
This work was funded in part by grant P40RR17447 from the National Institute of Health and award RP150637 from the Cancer Prevention and Research Institute of Texas. We would like to thank Dr. Darwin J. Prockop for his support on the project.
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 |
8-channel pipette, 10 – 100 µL | Eppendorf | 022453904 | preparation of hanging drops |
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-y (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, e.t.c.) |
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, 20G, 1 inch | Terumo | SR*FNP2025 | Delivery of shperoids into peritoneal cavity |
Sterile micropipette tips | Eppendorf | varies | liquid/cells handling |