異種非含有条件下での3次元培養における治療間葉系幹/間質細胞の生産と管理（MSC）スフェロイドプライム

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.