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.

<ol>
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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. 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The authors declare they have no competing financial interests.

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><tbody><tr><td>MEM-&amp;alpha; (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 &amp;micro;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&amp;nbsp;SIGMA</td><td>cell culture</td></tr><tr><td>Thermo Forma water-jacketed CO&lt;sub&gt;2&lt;/sub&gt; 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&amp;amp;M Health Science Center College of Medicine Institute for Regenerative Medicine at Scott &amp;amp; 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 &amp;deg;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&amp;nbsp;SIGMA</td><td>Component of xeno-free MSC media</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&amp;nbsp;</td><td>Qiagen</td><td>79254</td><td>On-column DNA elimination during total RNA extraction</td></tr><tr><td>&amp;beta;-mercaptoethanol&amp;nbsp;</td><td>Sigma-Aldrich</td><td>M6250&amp;nbsp;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 &amp;deg;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&amp;amp;D Systems</td><td>KGE004B</td><td>estimation of cytokine concentration in the sample</td></tr><tr><td>DMEM (Dulbecco&amp;rsquo;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>&lt;em&gt;in vitro&lt;/em&gt; macrophage stimulation</td></tr><tr><td>LPS (lipopolysaccharide)</td><td>Sigma-aldrich</td><td>L4130</td><td>&lt;em&gt;in vitro&lt;/em&gt; macrophage stimulation</td></tr><tr><td>Mouse TNF-a ELISA kit</td><td>R&amp;amp;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&amp;amp;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>&lt;em&gt;in vivo&lt;/em&gt; spheroid delivery; splenocyte preparation</td></tr><tr><td>Anti-Mouse CD3e Functional Grade Purified</td><td>eBioscience</td><td>145-2C11</td><td>&lt;em&gt;in vitro&lt;/em&gt; splenocyte stimulation</td></tr><tr><td>70 &amp;mu;m strainer&amp;nbsp;</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-&amp;#947; (interferon gamma) ELISA kit</td><td>R&amp;amp;D Systems</td><td>MIF 00</td><td>estimation of cytokine concentration in the sample</td></tr><tr><td>LNCaP prostate cancer cells&amp;nbsp;</td><td>ATCC</td><td>CRL-1740</td><td>study the effect of 3D MSCs on cancer cell lines &lt;em&gt;in vitro&lt;/em&gt;</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, &lt;em&gt;etc.&lt;/em&gt;)</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 &lt;em&gt;in vivo&lt;/em&gt; injections</td></tr><tr><td>Isoflurane</td><td>MWI Vet Supply</td><td>502017</td><td>Anesthesia for &lt;em&gt;in vivo&lt;/em&gt; 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, 20 G , 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 ...

production-administration-therapeutic-mesenchymal-stemstromal-cell

Research

JoVE Journal

Developmental Biology

11.8K Views.  University of Mary Hardin-Baylor. 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.

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

Production of Large Numbers of Size-controlled Tumor Spheroids Using Microwell Plates

Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically relevant than conventional adherent-sheet cultures, they more accurately recapitulate the complexity and interactions present in real tumors. In order to harness this model to better assess tumor biology, or the efficacy of novel therapeutic agents, it is necessary to be able to generate spheroids reproducibly, in a controlled manner and in significant numbers.
The AggreWell system consists of a high-density array of pyramid-shaped microwells, into which a suspension of single cells is centrifuged. The numbers of cells clustering at the bottom of each microwell, and the number and ratio of distinct cell types involved depend only on the properties of the suspension introduced by the experimenter. Thus, we are able to generate tumor spheroids of arbitrary size and composition without needing to modify the underlying platform technology. The hundreds of microwells per square centimeter of culture surface area in turn ensure that extremely high production levels may be attained via a straightforward, nonlabor-intensive process. We therefore expect that this protocol will be broadly useful to researchers in the tumor spheroid field.

We introduce a protocol for the generation of large numbers (thousands to hundreds of thousands) of uniform size- and composition-controlled tumor spheroids, using commercially available microwell plates.

Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically ...

Bioengineering

Formation of Human Periodontal Ligament Cell Spheroids on Chitosan Films

Periodontal ligament (PDL) cells hold great promise for periodontal tissue regeneration. Conventionally, PDL cells are cultured on two-dimensional (2D) substrates such as tissue culture polystyrene (TCPS). However, characteristic changes of PDL cells have been observed during in vitro culture. This phenomenon is probably because the 2D TCPS differs from the in vivo three-dimensional (3D) microenvironment. Compared to cells cultured on 2D substrates, cells grown in a 3D microenvironment exhibit more similarities to in vivo cells. Therefore, 3D cell culture models provide a promising alternative for conventional 2D monolayer cell culture. To improve conventional PDL cell culture models, we have recently developed a 3D cell culture method, which is based on spheroid formation of PDL cells on chitosan films. Here, we present detailed cell spheroid culture protocols based on chitosan films. The 3D culture system of PDL cellular spheroids overcome some of the limitations related to conventional 2D monolayer cell culture, and thus may be suitable for producing PDL cells with an enhanced therapeutic efficacy for future periodontal tissue regeneration.

Here, we present protocols of culturing human periodontal ligament (PDL) cell spheroids by chitosan films. The culture of three-dimensional (3D) cellular spheroids provides an alternative to conventional tissue culture polystyrene (TCPS) culture system.

Periodontal ligament (PDL) cells hold great promise for periodontal tissue regeneration. Conventionally, PDL cells are cultured on two-dimensional (2D) ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

Mesenchymal Stromal Cell Culture and Delivery in Autologous Conditions: A Smart Approach for Orthopedic Applications

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on potential biohazard and inflammation risks exerted by xenogeneic nutrients for their culture. Human derivatives or recombinant materials are the first choice candidates to reduce these reactions. Therefore, culture supplements and materials of autologous origin represent the best nutrients and the safest products.
Here, we describe a new protocol for the isolation and culture of bone marrow hMSCs in autologous conditions &#8212;&#160;namely,&#160;patient-derived serum as a supplement for the culture medium and fibrin as a scaffold for hMSC administration. Indeed, hMSC/fibrin clot constructs could be extremely useful for several clinical applications. In particular, we focus on their use in orthopedic surgery, where the fibrin clot derived from the donor&#39;s own blood allowed effective cell delivery and nutrient/waste exchanges. To ensure optimal safety conditions, it is of the utmost importance to avoid the risks of hMSC transformation and tissue overgrowth. For these reasons, the approach described in this paper also indicates a minimally ex vivo hMSC expansion, to reduce cell senescence and morphologic changes, and short-term osteo-differentiation before implantation, to induce osteogenic lineage specification, thus decreasing the risk of subsequent uncontrolled proliferation.

Culturing human Mesenchymal Stromal Cells (hMSCs) with autologous serum, reduces the risks of rejection by xenogeneic material and other negative effects. It also allows for the recovery of a subset of mesodermal progenitors, which can deliver fresh hMSCs. Embedding hMSCs in an autologous fibrin clot enables easy handling and effective surgical implantation.

Human Mesenchymal Stromal Cells (hMSCs) are cultured in vitro with different media. Limits on their use in clinical settings, however, mainly depend on ...

Generation of Tissue Spheroids via a 3D Printed Stamp-Like Device

Advances in 3D cell culture have developed more physiologically relevant in vitro models, such as tissue spheroids. Cells cultivated as spheroids have more realistic biological responses that resemble the in vivo environment. Due to their advantages, tissue spheroids represent an emerging trend toward superior, more reliable, and more predictive study models with a broad range of biotechnological applicability. However, reproducible platforms that can achieve large-scale production of tissue spheroids have become an unmet need in fully exploring and boosting their potential. Herein, the large-scale production of homogeneous tissue spheroids is reported using a low-cost and time-effective methodology. A 3D printed stamp-like device is developed to generate up to 4,716 spheroids per 6-well plate. The device is fabricated by the stereolithography method using a photocurable resin. The final device is composed of cylindrical micropins, with a height of 1.3 mm and a width of 650 &#181;m. This approach allows the fast generation of homogeneous spheroids and co-cultured spheroids with uniform shape and size and &gt;95% cell viability. Moreover, the stamp-like device is tunable for different sizes of well plates and Petri dishes. It is easily sterilized and can be reused for long periods. The efficient large-scale production of homogeneous tissue spheroids is essential to leverage their translation for multiple areas of industry, such as tissue engineering, drug development, disease modeling, and on-demand personalized medicine.

Generation of Tissue Spheroids via a 3D Printed Stamp-Like Device

The present protocol describes a technique to produce tissue spheroids on a large scale cost-effectively using a 3D printed stamp-like device.

Advances in 3D cell culture have developed more physiologically relevant in vitro models, such as tissue spheroids. Cells cultivated as spheroids have ...

Biochemistry

A Label-free Xenograft Model for Investigating the Behavior of Human Stem Cell Spheroids in Chick Embryos

Xenografts are valuable methods to investigate the behavior of human cells in vivo. In particular, the embryonic environment provides cues for cell migration, differentiation, and morphogenesis, with unique instructive signals and germ layer identity that are often absent from adult xenograft models. In addition, embryonic models cannot discriminate self versus non-self tissues, eliminating the risk of rejection of the graft and the need for immune suppression of the host. This paper presents a methodology for transplantation of spheroids of human cells into chicken embryos, which are accessible, amenable to manipulation, and develop at 37 &#176;C. 
Spheroids allow the selection of a specific region of the embryo for transplantation. After being grafted, the cells become integrated into the host tissue, allowing the follow-up of their migration, growth, and differentiation. This model is flexible enough to allow the utilization of different adherent populations, including heterogeneous primary cell populations and cancer cells. To circumvent the need for prior cell labeling, a protocol for the identification of donor cells through hybridization of human-specific Alu probes is also described, which is particularly important when investigating heterogeneous cell populations. Furthermore, DNA probes can be easily adapted to identify other donor species. This protocol will describe the general methods for preparing spheroids, grafting into chicken embryos, fixing and processing tissue for paraffin sectioning, and finally identifying the human cells using DNA in situ hybridization. Suggested controls, examples of interpretation of results and various cell behaviors that can be assayed will be discussed in addition to the limitations of this method.

Here, we describe a method to transplant and identify human cell spheroids into chick embryos. This xenograft model uses the embryonic microenvironment as a source of instructive signals to assay cell migration, differentiation, and tropism and is especially suited for the study of primary and/or heterogeneous cell populations.

Xenografts are valuable methods to investigate the behavior of human cells in vivo. In particular, the embryonic environment provides cues for cell ...

3D-PDAC Model Capturing Desmoplasia for Therapeutic Evaluation

Growing Desmoplastic Three-Dimensional Pancreatic Cancer Spheroids from Co-Culture

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers with a 5-year survival rate of &#60;12%. The biggest barrier to therapy is the dense desmoplastic extracellular matrix (ECM) that surrounds the tumor and reduces vascularization, generally termed desmoplasia. A variety of drug combinations and formulations have been tested to treat the cancer, and although many of them show success pre-clinically, they fail clinically. It, therefore, becomes important to have a clinically relevant model available that can predict the response of the tumor to therapy. This model has been previously validated against resected clinical tumors. Here a simple protocol to grow desmoplastic three-dimensional (3D)-coculture spheroids is described that can naturally generating a robust ECM and do not require any external matrix sources or scaffold to support their growth.
Briefly human pancreatic stellate cells (HPaSteC) and PANC-1 cells are used to prepare a suspension containing the cells in a 1:2 ratio, respectively. The cells are plated in a poly-HEMA coated, 96-well low attachment U-well plate. The plate is centrifuged to allow the cells to form an initial pellet. The plate is stored in the incubator at 37 &#176;C with 5% CO2, and media is replaced every 3 days. Plates can be imaged at designated intervals to measure spheroid volume. Following 14 days of culture, mature desmoplastic spheroids are formed (i.e. average volume of 0.048 + 0.012 mm3&#160;(451 &#181;m x 462.84 &#181;m)) and can be utilized for experimental therapy assessment. Mature ECM components include collagen-I, hyaluronic acid, fibronectin, and laminin.

Author Spotlight: Validating Cancer Therapy Responses with Desmoplastic Spheroid Models

Pancreatic cancer remains one of the toughest cancers to treat. Therefore, it is critical that pre-clinical models evaluating treatment efficacy are reproducible and clinically relevant. This protocol describes a simple co-culture procedure to generate reproducible, clinically relevant desmoplastic spheroids.

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers with a 5-year survival rate of &#60;12%. The biggest barrier to therapy is the ...

Cancer Research

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches.
Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method.
The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

Here, we describe the large-scale production of adipose-derived stromal/stem cell (ASC) spheroids using an automated pipetting system to seed the cell suspension, thus ensuring homogeneity of spheroid size and shape. These ASC spheroids can be used as building blocks for 3D bioprinting approaches.

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue ...

Isolation and Culture of Human Adipose-Derived Stem Cells With an Innovative Xenogeneic-Free Method for Human Therapy

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission due to the introduction of animal-derived products during in vitro manipulation. The xenogeneic components, such as collagenase or fetal bovine serum, commonly used during the cell isolation and expansion steps could be associated with the potential risks of immune reactivity or viral, bacterial, and prion infection in the receiving patients. Following good manufacturing practice guidelines, chemical tissue dissociation should be avoided, while fetal bovine serum (FBS) can be substituted with xenogeneic-free supplements. Moreover, to ensure the safety of cell products, the definition of more reliable and reproducible methods is important. We have developed an innovative, completely xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells without altering their properties compared to collagenase FBS-cultured standard protocols. Here, human adipose-derived stem cells (hASCs) were isolated from abdominal adipose tissue. The sample was mechanically minced with scissors/a scalpel, micro-dissected and mechanically dispersed in a 10 cm Petri dish, and prepared with scalpel incisions to facilitate the attachment of the tissue fragments and the migration of hASCs. Following the washing steps, hASCs were selected due to their plastic adherence without enzymatic digestion. The isolated hASCs were cultured with medium supplemented with 5% heparin-free human platelet lysate and detached with an animal-free trypsin substitute. Following good manufacturing practice (GMP) directions on the production of cell products intended for human therapy, no antibiotics were used in any culture media.

Xenogeneic (chemical or animal-derived) products introduced in the cell therapy preparation/manipulation steps are associated with an increased risk of immune reactivity and pathogenic transmission in host patients. Here, a complete xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells is described.

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission ...

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

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