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.

1. MSC Isolation and Expansion
<ol>
	<li>Obtain early passage MSCs from Center for the Preparation and Distribution of Adult Stem Cells (<a href="http://medicine.tamhsc.edu/irm/msc-distribution.html" target="_blank">http://medicine.tamhsc.edu/irm/msc-distribution.html</a>)15 as frozen vials. Alternatively, isolate MSCs from bone marrow aspirates following a routine protocol14 and store as frozen vials.</li>
	<li>Prepare complete culture medium (CCM), which is minimal esse...

<ol>
	<li>Keating, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stromal+cells:+New+directions.">Mesenchymal stromal cells: New directions.</a> Cell Stem Cell. 10 (6), 709-716 (2012).</li><li>English, K., Wood, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stromal+cells+in+transplantation+rejection+and+tolerance.">Mesenchymal stromal cells in transplantation rejection and tolerance.</a> Cold Spring Harb Perspect.Med. 3 (5), a015560 (2013).</li><li>Le Blanc, K., Mougiakakos, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotent+mesenchymal+stromal+cells+and+the+innate+immune+system.">Multipotent mesenchymal stromal cells and the innate immune system.</a> Nat.Rev.Immunol. 12 (5), 383-396 (2012).</li><li>Follin, B., Juhl, M., Cohen, S., Perdersen, A. E., Kastrup, J., Ekblond, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+Paracrine+Immunomodulatory+Potential+of+Mesenchymal+Stromal+Cells+in+Three-Dimensional+Culture.">Increased Paracrine Immunomodulatory Potential of Mesenchymal Stromal Cells in Three-Dimensional Culture.</a> Tissue Eng.Part B.Rev. , (2016).</li><li>Cesarz, Z., Tamama, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spheroid+Culture+of+Mesenchymal+Stem+Cells.">Spheroid Culture of Mesenchymal Stem Cells.</a> Stem Cells Int. , 9176357 (2016).</li><li>Achilli, T. M., Meyer, J., Morgan, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+the+formation,+use+and+understanding+of+multi-cellular+spheroids.">Advances in the formation, use and understanding of multi-cellular spheroids.</a> Expert Opin.Biol. Ther. 12 (10), 1347-1360 (2012).</li><li>Sart, S., Tsai, A. C., Li, Y., Ma, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+aggregates+of+mesenchymal+stem+cells:+cellular+mechanisms,+biological+properties,+and+applications.">Three-dimensional aggregates of mesenchymal stem cells: cellular mechanisms, biological properties, and applications.</a> Tissue Eng.Part B.Rev. 20 (5), 365-380 (2014).</li><li>Bartosh, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aggregation+of+human+mesenchymal+stromal+cells+(MSCs)+into+3D+spheroids+enhances+their+antiinflammatory+properties.">Aggregation of human mesenchymal stromal cells (MSCs) into 3D spheroids enhances their antiinflammatory properties.</a> Proc.Natl.Acad.Sci.U.S.A. 107 (31), 13724-13729 (2010).</li><li>Potapova, I. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+support+migration,+extracellular+matrix+invasion,+proliferation,+and+survival+of+endothelial+cells+in+vitro.">Mesenchymal stem cells support migration, extracellular matrix invasion, proliferation, and survival of endothelial cells in vitro.</a> Stem Cells. 25 (7), 1761-1768 (2007).</li><li>Frith, J. E., Thomson, B., Genever, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+three-dimensional+culture+methods+enhance+mesenchymal+stem+cell+properties+and+increase+therapeutic+potential.">Dynamic three-dimensional culture methods enhance mesenchymal stem cell properties and increase therapeutic potential.</a> Tissue Eng.Part C.Methods. 16 (4), 735-749 (2010).</li><li>Lee, R. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravenous+hMSCs+Improve+Myocardial+Infarction+in+Mice+because+Cells+Embolized+in+Lung+Are+Activated+to+Secrete+the+Anti-inflammatory+Protein+TSG-6.">Intravenous hMSCs Improve Myocardial Infarction in Mice because Cells Embolized in Lung Are Activated to Secrete the Anti-inflammatory Protein TSG-6.</a> Cell Stem Cell. 5 (1), 54-63 (2009).</li><li>Bartosh, T. J., Ylostalo, J. H., Bazhanov, N., Kuhlman, J., Prockop, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+compaction+of+human+mesenchymal+stem/precursor+cells+into+spheres+self-activates+caspase-dependent+il1+signaling+to+enhance+secretion+of+modulators+of+inflammation+and+immunity+(PGE2,+TSG6,+and+STC1).">Dynamic compaction of human mesenchymal stem/precursor cells into spheres self-activates caspase-dependent il1 signaling to enhance secretion of modulators of inflammation and immunity (PGE2, TSG6, and STC1).</a> Stem Cells. 31 (11), 2443-2456 (2013).</li><li>Ylostalo, J. H., Bartosh, T. J., Coble, K., Prockop, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+mesenchymal+stem/stromal+cells+cultured+as+spheroids+are+self-activated+to+produce+prostaglandin+E2+that+directs+stimulated+macrophages+into+an+anti-inflammatory+phenotype.">Human mesenchymal stem/stromal cells cultured as spheroids are self-activated to produce prostaglandin E2 that directs stimulated macrophages into an anti-inflammatory phenotype.</a> Stem Cells. 30 (10), 2283-2296 (2012).</li><li>Ylostalo, J. H., Bartosh, T. J., Tiblow, A., Prockop, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unique+characteristics+of+human+mesenchymal+stromal/progenitor+cells+pre-activated+in+3-dimensional+cultures+under+different+conditions.">Unique characteristics of human mesenchymal stromal/progenitor cells pre-activated in 3-dimensional cultures under different conditions.</a> Cytotherapy. 16 (11), 1486-1500 (2014).</li><li>Pittenger, M. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multilineage+potential+of+adult+human+mesenchymal+stem+cells.">Multilineage potential of adult human mesenchymal stem cells.</a> Science. 284 (5411), 143-147 (1999).</li><li>Uccelli, A., Moretta, L., Pistoia, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+in+health+and+disease.">Mesenchymal stem cells in health and disease.</a> Nat.Rev.Immunol. 8 (9), 726-736 (2008).</li><li>Dominici, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Minimal+criteria+for+defining+multipotent+mesenchymal+stromal+cells.+The+International+Society+for+Cellular+Therapy+position+statement.">Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement.</a> Cytotherapy. 8 (4), 315-317 (2006).</li><li>Fontaine, M. J., Shih, H., Schafer, R., Pittenger, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unraveling+the+Mesenchymal+Stromal+Cells'+Paracrine+Immunomodulatory+Effects.">Unraveling the Mesenchymal Stromal Cells' Paracrine Immunomodulatory Effects.</a> Transfus.Med.Rev. 30 (1), 37-43 (2016).</li><li>Nemeth, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+marrow+stromal+cells+attenuate+sepsis+via+prostaglandin+E(2)-dependent+reprogramming+of+host+macrophages+to+increase+their+interleukin-10+production.">Bone marrow stromal cells attenuate sepsis via prostaglandin E(2)-dependent reprogramming of host macrophages to increase their interleukin-10 production.</a> Nat.Med. 15 (1), 42-49 (2009).</li><li>Prockop, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+review:+two+negative+feedback+loops+place+mesenchymal+stem/stromal+cells+at+the+center+of+early+regulators+of+inflammation.">Concise review: two negative feedback loops place mesenchymal stem/stromal cells at the center of early regulators of inflammation.</a> Stem Cells. 31 (10), 2042-2046 (2013).</li><li>Krampera, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stromal+cell+'licensing':+a+multistep+process.">Mesenchymal stromal cell 'licensing': a multistep process.</a> Leukemia. 25 (9), 1408-1414 (2011).</li><li>Saleh, F. A., Genever, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turning+round:+multipotent+stromal+cells,+a+three-dimensional+revolution?.">Turning round: multipotent stromal cells, a three-dimensional revolution?.</a> Cytotherapy. 13 (8), 903-912 (2011).</li><li>Tsai, A. C., Liu, Y., Yuan, X., Ma, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compaction,+fusion,+and+functional+activation+of+three-dimensional+human+mesenchymal+stem+cell+aggregate.">Compaction, fusion, and functional activation of three-dimensional human mesenchymal stem cell aggregate.</a> Tissue Eng.Part A. 21 (9-10), 1705-1719 (2015).</li><li>Yeh, H. Y., Liu, B. H., Hsu, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+calcium-dependent+regulation+of+spheroid+formation+and+cardiomyogenic+differentiation+for+MSCs+on+chitosan+membranes.">The calcium-dependent regulation of spheroid formation and cardiomyogenic differentiation for MSCs on chitosan membranes.</a> Biomaterials. 33 (35), 8943-8954 (2012).</li><li>Lee, E. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spherical+bullet+formation+via+E-cadherin+promotes+therapeutic+potency+of+mesenchymal+stem+cells+derived+from+human+umbilical+cord+blood+for+myocardial+infarction.">Spherical bullet formation via E-cadherin promotes therapeutic potency of mesenchymal stem cells derived from human umbilical cord blood for myocardial infarction.</a> Mol.Ther. 20 (7), 1424-1433 (2012).</li></ol>

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...

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 &#34;lost/diminished&#34; 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 &#34;hit and run&#34;. 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>MEM-&#945; (minimal essential medium alpha)</td>
			<td>ThermoFisher/Gibco</td>
			<td>12561049; 12561056; 12561072</td>
			<td>minimal essential medium for preparation of MSC growth medium (CCM)</td>
		</tr>
		<tr>
			<td>FBS (fetal bovine serum), premium select</td>
			<td>Atlanta Biologicals</td>
			<td>S11595; S11510; S11550; S11595-24</td>
			<td>component of complete culture media for all types of cells</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>ThermoFisher/Gibco</td>
			<td>25030081; 25030149; 25030164</td>
			<td>component of complete culture media for all types of cells</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>ThermoFisher/Gibco</td>
			<td>15070063</td>
			<td>component of complete culture media for all types of cells</td>
		</tr>
		<tr>
			<td>Sterilization Filter Units, 0.22 &#181;m PES membrane</td>
			<td>MilliporeSigma</td>
			<td>SCGPU01RE; SCGPU02RE; SCGPU05RE; SCGPU10RE; SCGPU11RE</td>
			<td>media sterilization</td>
		</tr>
		<tr>
			<td>150 mm cell culture dish</td>
			<td>Nunc</td>
			<td>D8554&#160;SIGMA</td>
			<td>cell culture</td>
		</tr>
		<tr>
			<td>Thermo Forma water-jacketed CO2 humidified incubator</td>
			<td>Thermo Fisher</td>
			<td>Model 3110</td>
			<td>incubation of cultured cells</td>
		</tr>
		<tr>
			<td>Early passage MCSs</td>
			<td>Center for the preparation and Distribution of Adult Stem Cells at The Texas A&#38;M Health Science Center College of Medicine Institute for Regenerative Medicine at Scott &#38; White</td>
			<td>NA</td>
			<td>preparation of 2D and 3D cultures of MSCs</td>
		</tr>
		<tr>
			<td>water bath</td>
			<td>VWR</td>
			<td>89501-468</td>
			<td>warming media to 37 &#176;C</td>
		</tr>
		<tr>
			<td>Pipettes</td>
			<td>Eppendorf</td>
			<td>492000904</td>
			<td>manual liquid handling</td>
		</tr>
		<tr>
			<td>Pipete-Aid</td>
			<td>Drummond Scientific Company</td>
			<td>4-000-300</td>
			<td>handling sereological pipetes</td>
		</tr>
		<tr>
			<td>Costar sterile serological pipet (5, 10, 25 and 50 ml)</td>
			<td>Corning</td>
			<td>4487; 4101; 4251; 4490</td>
			<td>liquid handling</td>
		</tr>
		<tr>
			<td>PBS (phosphate buffered saline), pH 7.4</td>
			<td>ThermoFisher/Gibco</td>
			<td>10010023; 10010072; 10010031; 10010049</td>
			<td>cell culture processing</td>
		</tr>
		<tr>
			<td>0.25% trypsin/EDTA solution</td>
			<td>ThermoFisher/Gibco</td>
			<td>25200056; 25200072; 25200114</td>
			<td>lifting adherent cells and dispersing cell aggregates</td>
		</tr>
		<tr>
			<td>15 ml conical tube</td>
			<td>Corning/BD Falcon</td>
			<td>352097</td>
			<td>cell centrifugation</td>
		</tr>
		<tr>
			<td>50 ml conical tube</td>
			<td>Corning/BD Falcon</td>
			<td>352098</td>
			<td>cell centrifugation</td>
		</tr>
		<tr>
			<td>Eppendor refrigerated centrifuge</td>
			<td>Eppendorf/Fisher Scientific</td>
			<td>Model 5810R</td>
			<td>cell centrifugation</td>
		</tr>
		<tr>
			<td>hemocytometer</td>
			<td>Fisher Scientific</td>
			<td>26716</td>
			<td>cell counting</td>
		</tr>
		<tr>
			<td>trypan blue</td>
			<td>Sigma-Aldrich</td>
			<td>T8154 SIGMA</td>
			<td>dead cell exclusion during cell counting in hemacytometer</td>
		</tr>
		<tr>
			<td>Defined xenofree MSC medium-1 (XFM-1)</td>
			<td>ThermoFisher/Gibco</td>
			<td>A1067501</td>
			<td>Xeno-free media specifically formulated for the growth and expansion of human mesenchymal stem cells</td>
		</tr>
		<tr>
			<td>Defined xenofree MSC medium-2 (XFM-2)</td>
			<td>Stem Cell Technologies</td>
			<td>5420</td>
			<td>Defined, xeno-free medium for human mesenchymal stem cells</td>
		</tr>
		<tr>
			<td>HSA (Human serum albumin)</td>
			<td>Gemini</td>
			<td>800-120</td>
			<td>Component of xeno-free MSC media</td>
		</tr>
		<tr>
			<td>rHSA (recombinant Human serum albumin)</td>
			<td>Sigma-Aldrich</td>
			<td>A9731&#160;SIGMA</td>
			<td>Component of xeno-free MSC media</td>
		</tr>
		<tr>
			<td>8-channel pipette, 10&#8239;&#8211;&#8239;100&#8239;&#181;L</td>
			<td>Eppendorf</td>
			<td>022453904</td>
			<td>preparation of hanging drops</td>
		</tr>
		<tr>
			<td>Total RNA isolation Mini Kit</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td>Total RNA extraction</td>
		</tr>
		<tr>
			<td>Qiashredder</td>
			<td>Qiagen</td>
			<td>79654</td>
			<td>Sample homogenization prior to total RNA extraction</td>
		</tr>
		<tr>
			<td>RNAse-free DNase Set&#160;</td>
			<td>Qiagen</td>
			<td>79254</td>
			<td>On-column DNA elimination during total RNA extraction</td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>M6250&#160;ALDRICH</td>
			<td>inhibition of RNAses in RLT buffer</td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>VWR</td>
			<td>97043-562</td>
			<td>mixing sample</td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Biorad</td>
			<td>NA</td>
			<td>RNA concentration and quality</td>
		</tr>
		<tr>
			<td>High capacity cDNA Reverse Transcription Kit</td>
			<td>ThermoFisher/Applied Biosystems</td>
			<td>4368814</td>
			<td>transcription of total RNA into cDNA</td>
		</tr>
		<tr>
			<td>Gene Expression Assays</td>
			<td>ThermoFisher/Applied Biosystems</td>
			<td>varies</td>
			<td>primer/probe combination for real-time PCR</td>
		</tr>
		<tr>
			<td>Fast Universal PCR Master Mix</td>
			<td>ThermoFisher/Applied Biosystems</td>
			<td>4352042; 4364103; 4366073; 4367846</td>
			<td>master mix for real-time PCR reaction</td>
		</tr>
		<tr>
			<td>Real-time PCR system (ABI Prism 7900 HT Sequence Detection System)</td>
			<td>ABI Prizm</td>
			<td>NA</td>
			<td>real-time PCR</td>
		</tr>
		<tr>
			<td>1.5 ml centrifuge tube</td>
			<td>Eppendorf</td>
			<td>22364111</td>
			<td>cell centrifugation, sample collection and storage</td>
		</tr>
		<tr>
			<td>(-80&#176;C) freezer</td>
			<td>Thermo Fisher</td>
			<td>Model Thermo Forma 8695</td>
			<td>sample storage</td>
		</tr>
		<tr>
			<td>PGE2 (Prostaglandin E2) ELISA Kit</td>
			<td>R&#38;D Systems</td>
			<td>KGE004B</td>
			<td>estimation of cytokine concentration in the sample</td>
		</tr>
		<tr>
			<td>DMEM (Dulbecco&#8217;s modified Eagle medium)</td>
			<td>ThermoFisher/Gibco</td>
			<td>10566-016; 10566-024;10566-032</td>
			<td>macrophage culture media</td>
		</tr>
		<tr>
			<td>J774 mouse macrophages</td>
			<td>ATCC</td>
			<td>TIB-67</td>
			<td>mouse macrophage cell line</td>
		</tr>
		<tr>
			<td>12-well plate</td>
			<td>Corning</td>
			<td>3513</td>
			<td>in-vitro macrophage stimulation</td>
		</tr>
		<tr>
			<td>LPS (lipopolysaccharide)</td>
			<td>Sigma-aldrich</td>
			<td>L4130</td>
			<td>in vitro macrophage stimulation</td>
		</tr>
		<tr>
			<td>Mouse TNF-a ELISA kit</td>
			<td>R&#38;D Systems</td>
			<td>MTA00B</td>
			<td>estimation of cytokine concentration in the sample</td>
		</tr>
		<tr>
			<td>Mouse IL-10 (interleukin 10) ELISA kit</td>
			<td>R&#38;D Systems</td>
			<td>M1000B</td>
			<td>estimation of cytokine concentration in the sample</td>
		</tr>
		<tr>
			<td>RPMI-1640 medium</td>
			<td>ThermoFisher/Gibco</td>
			<td>11875-085</td>
			<td>splenocyte culture media</td>
		</tr>
		<tr>
			<td>BALB/c mice</td>
			<td>The Jackson Laboratory</td>
			<td>651</td>
			<td>in vivo spheroid delivery; splenocyte preparation</td>
		</tr>
		<tr>
			<td>Anti-Mouse CD3e Functional Grade Purified</td>
			<td>eBioscience</td>
			<td>145-2C11</td>
			<td>In vitro splenocyte stimulation</td>
		</tr>
		<tr>
			<td>70 &#956;m strainer&#160;</td>
			<td>Corning</td>
			<td>352350</td>
			<td>Splenocyte preparation</td>
		</tr>
		<tr>
			<td>Red blood cell lysis solution (1x)</td>
			<td>Affymetrix eBioscience</td>
			<td>00-4333</td>
			<td>removal of red blood cells during splenocyte isolation</td>
		</tr>
		<tr>
			<td>Mouse IFN-y (interferon gamma) ELISA kit</td>
			<td>R&#38;D Systems</td>
			<td>MIF 00</td>
			<td>estimation of cytokine concentration in the sample</td>
		</tr>
		<tr>
			<td>LNCaP prostate cancer cells&#160;</td>
			<td>ATCC</td>
			<td>CRL-1740</td>
			<td>study the effect of 3D MSCs on cancer cell lines in vitro</td>
		</tr>
		<tr>
			<td>DNA-based cell proliferation assay kit</td>
			<td>ThermoFisher</td>
			<td>C7026</td>
			<td>cell number measurement based on DNA content</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S5150</td>
			<td>component of lysis reagent</td>
		</tr>
		<tr>
			<td>EDTA (ethylenediaminetetraacetic acid)</td>
			<td>ThermoFisher</td>
			<td>FERR1021</td>
			<td>calcium chelator, component of lysis reagent</td>
		</tr>
		<tr>
			<td>Rnase A</td>
			<td>Qiagen</td>
			<td>19101</td>
			<td>RNA degradation for measurement of DNA</td>
		</tr>
		<tr>
			<td>Filter-based multi-mode microplate reader</td>
			<td>BMG Technology</td>
			<td>NA</td>
			<td>Microplate assays (ELISA, cell quantification, e.t.c.)</td>
		</tr>
		<tr>
			<td>HBSS (Hanks balanced salt solution), no calcium, no magnesium, no phenol red</td>
			<td>ThermoFisher/Gibco</td>
			<td>14175079</td>
			<td>resupsension of MSC spheroids prior to in vivo injections</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>MWI Vet Supply</td>
			<td>502017</td>
			<td>Anesthesia for in vivo injections</td>
		</tr>
		<tr>
			<td>Oxygen, compressed gas</td>
			<td>Praxair</td>
			<td>NA</td>
			<td>For use with isoflurane</td>
		</tr>
		<tr>
			<td>Thermo Forma BSL-2 cabinet</td>
			<td>Thermo Fisher</td>
			<td>Model 1385</td>
			<td>Sterile cell culture</td>
		</tr>
		<tr>
			<td>Safety I.V. catheter/needle stiletto, 20G, 1 inch</td>
			<td>Terumo</td>
			<td>SR*FNP2025</td>
			<td>Delivery of shperoids into peritoneal cavity</td>
		</tr>
		<tr>
			<td>Sterile micropipette tips</td>
			<td>Eppendorf</td>
			<td>varies</td>
			<td>liquid/cells handling</td>
		</tr>
	</tbody>
</table>

production and administration of therapeutic mesenchymal stem/stromal cell (msc) spheroids primed in 3-d cultures under xeno-free conditions

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 &#39;spheroids&#39; 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.

In the current work, hanging drop cultures were employed to generate compact spherical micro-tissues or &#39;spheroids&#39; 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...

Watch this Scientific Journal Video about Production and Administration of Therapeutic Mesenchymal Stem/Stromal Cell MSC Spheroids Primed in 3-D Cultures Under Xeno-free Conditions at JoVE.com

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

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.

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

Mesenchymal stem/stromal cells (MSCs) hold great promise in bioengineering and regenerative medicine. MSCs can be isolated from multiple adult tissues via ...

The overall goal of these experiments is to efficiently generate and inject therapeutic spherical aggregates, or spheroids, of mesenchymal stem/stromal cells, or MSCs, primed under xeno-free conditions in experimental and clinical applications. These methods can help overcome major barriers in the field of MSC therapeutics, such as poor cell retention, survival and functionality. That's often seen following transplantation of dispersed cells and to adverse conditions commonly associated with tissue injury.The main advantage of these procedures is that the MSC spheroids can be prepared, primed and injected in the absence of immunogenic animal products, such as FBS, which currently limits some clinical applications of the cells and can also confound interpretation of some pre-clinical data. Although this mechanism is potentially very beneficial in mediating MSC-based efficacy, it can also be used for understanding MSC mediated tissue repair and also how the other media formulations can change the therapeutic genex plus. Demonstrating these procedures will be Josh Beaver and Bret Clough, two technicians from my laboratory.After seeding and incubating mesenchymal stem cells, according to the text protocol, use trips and EDTA harvest the cells following centrifugation of harvested cells at 450 times G, combine all the cells into a single conical tube with the appropriate medium. Centrifuge the cells and re-suspend the pellet in a small volume of medium. Then after counting the cells, to generate spheroids of approximately 25, 000 cells, dilute the harvested MSCs in one of the following medias at 714 cells per microliter.Next, pipette 35 microliter drops of the cells onto the underside of a 150 millimeter inverted culture dish lid. Transfer 20 milliliters of room temperature PBS into the base of the dish and in one continuous steady motion, flip the lid containing the drops so that the drop apices face downward. Then carefully place the lid on the base of the culture dish.Transfer the dish to the incubator for three days without disturbing it to permit appropriate sphere assembly. Following the incubation, begin spheroid harvest by carefully removing the lid and inverting it so that the drops again face upward. Then angle the lid to approximately 10 to 20 degrees and use a cell lifter to push the drops containing the spheroids to the edge of the plate.Using a one milliliter pipette, transfer the sphere's end medium into a 15 milliliter conical tube and use room temperature PBS and the same pipette tip to wash the plate to ensure maximum recovery of spheroids. For real-time PCR assays, centrifuge the sphere suspension at 450 times G at room temperature for five minutes. Then aspirate the supernatant.With PBS, wash the spheroids and repeat both the centrifugation and aspiration steps. For delivery into animals, allow the spheres to settle to the bottom of the tube without centrifugation. To collect conditioned medium, or CM for assays of immunomodulatory and anti-cancer potential using a cell lifter and pipette as just demonstrated, harvest the spheroids and CM medium into a 15 milliliter conical tube.However, do not wash the lids with PBS. Following centrifugation at 450 times G for 5 to 10 minutes, transfer the supernatant into 1.5 milliliter tubes. Centrifuge the supernatant at 10, 000 times G and room temperature for 5 to 10 minutes.Then use a pipette to carefully collect the clarified CM into new 1.5 milliliter tubes for storage at minus 80 degrees Celsius. After preparing spheroids as just demonstrated in this video and waiting three to four minutes for them to settle to the bottom of the tube, aspirate the supernatant and wash the spheroids by adding HBSS supplemented with 0.2 to 0.5%HSA to maintain xeno-free conditions. Next, aspirate the supernatant.Overlay the spheroids with 100 to 200 microliters of sterile HBSS/HSA and place the tubes on ice. After anesthetizing a mouse according to the text protocol, place the animal inside the BSL-2 cabinet, dorsal aspect to down and use 70%ethanol to disinfect the lower mouse abdomen. Remove the cap of a 20-gauge intravenous catheter and while holding the mouse skin with one hand, locate the middle of an artificial line connecting a flexed knee joint and the genital area and with the needle tip of the catheter stiletto assembly pointing towards the midline, use the other hand to perform an intraperitoneal injection.Gently remove the metal stiletto needle from the catheter stiletto assembly. Check the stiletto for blood, urine and feces and discard it. Confirm proper placement of the plastic catheter by inserting a 100 to 200 microliter pipette tip with 100 microliters of sterile HBSS/HSA into the catheter syringe adapter forming a tight seal.Then slowly inject the fluid inside the peritoneal cavity. With a 100 to 200 microliter pipette tip, collect the MSA spheroids and HBSS/HSA and transfer the entire volume into the peritoneal cavity through the catheter as just demonstrated. Next, with 100 microliters of HBSS/HSA, wash the tube containing the spheres and transfer the entire volume of spheres into the peritoneal cavity.Place a gauze pad soaked in antiseptic over the catheter and slowly remove the catheter from the peritoneal cavity and discard it. Inspect the catheter for any remaining spheroids. Then gently massage the peritoneal cavity with the fingers to ensure even distribution of the spheroids.Immediately after delivering spheroids in the mice, evaluate the activation level of spheroids prepared for injections by transferring 40 to 120 spheres to the catheter into a 12 or 6 well plate containing one to two milliliters of Alpha MEM supplemented with 2%FBS and one X penicillin streptomycin. Six hours later, transfer the medium from the wells into 1.5 or 15 milliliter tubes and centrifuge the tubes 450 times G and room temperature for five minutes. Finally, transfer the clarified CM into new 1.5 milliliter tubes and use a commercial ELISA kit according to the text protocol to assay for PGE-2 concentration.As seen here, when approximately 25, 000 MSCs are suspended in 35 microliter drops of CCM, the cells first assemble into small aggregates, but eventually coalesce to generate a single compact sphere at 72 hours in the apex of the drop. This figure demonstrates that the formation of single compact spheres and XFM-1 requires supplementation with 13 milligrams per milliliter HSA, a concentration that reflects the estimated total protein content in CCM. During sphere assembly, the MSC phenotype changes radically and when in the appropriately chemically defined xeno-free medium, expression of numerous anti-inflammatory factors, including PGE2 and TSG6, is up-regulated.However, production of TSG6 and PGE2 is not markedly augmented in MSCs cultured as spheres in XFM-2 or XFM-1 without HSA. Finally, similar to the in vitro effects, XFM-1 HSA spheres injected into the peritoneal cavity of mice, immediately following induction of systemic inflammation by the IV administration of LPS, enhanced PGE2 and IL-10 levels in the peritoneum while decreasing proinflammatory TNF alpha. Once mastered, each plate of hanging drops can be prepared in one to two minutes.And the intraperitoneal delivery of in tact spheroids can be completed in 5 to 10 minutes per animal when performed properly. When producing MSC spheroids using hanging drop technique, it's important to remember that different xeno-free media formulations are not created equal in regards to sphere formation and enhancing the expression of therapeutic genes. Following this procedure, other methods like confocal microscopy can be used to understand the organization of the MSCs in the spheroids and also what might be some additional signaling pathways activated in the MSCs in different parts of the spheroid.After this development, this method allowed the MSC scientists to study other 3D culture conditions and how they affect MSC priming and therapeutic gene expression. After watching this video, you should have a good understanding of how to prime MSCs as spheroids using hanging drop technique, how to evaluate their therapeutic efficacy in vitro, and how to effectively deliver in tact spheroids into mice for in vivo efficacy testing. And don't forget that working with adult human stem cells require special equipment and training and can be hazardous if the cells are not handled properly.Appropriate PPE must be worn at all times when handling adult human stem cells.

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JoVE Journal

Developmental Biology

11.7K vistas.  University of Mary Hardin-Baylor. The overall goal of these experiments is to efficiently generate and inject therapeutic spherical aggregates, or spheroids, of mesenchymal stem/stromal cells, or MSCs, primed under xeno-free conditions in experimental and clinical applications. These methods can help overcome major barriers in the field of MSC therapeutics, such as poor cell retention, survival and functionality. That's often seen following transplantation of dispersed cells and to adverse conditions commonly associated with ...