The achievements of cell-based therapeutics have galvanized efforts to bring cell therapies to the market. To address the demands of the clinical and eventual commercial-scale production of cells, and with the increasing generation of large clinical datasets from chimeric antigen receptor T-cell immunotherapy, from transplants of engineered haematopoietic stem cells and from other promising cell therapies, an emphasis on biomanufacturing requirements becomes necessary. Robust infrastructure should address current limitations in cell harvesting, expansion, manipulation, purification, preservation and formulation, ultimately leading to successful therapy administration to patients at an acceptable cost. In this Review, we highlight case examples of cutting-edge bioprocessing technologies that improve biomanufacturing efficiency for cell therapies approaching clinical use.

Wound healing is a hierarchical process of intracellular and intercellular signaling. Insulin is a potent chemoattractant and mitogen for cells involved in wound healing. Insulin's potential to promote keratinocyte growth and stimulate collagen synthesis in fibroblasts is well described. However, there currently lacks an appropriate delivery mechanism capable of consistently supplying a wound environment with insulin; current approaches require repeated applications of insulin, which increase the chances of infecting the wound. In this study, we present a novel cell-based therapy that delivers insulin to the wound area in a constant or glucose-dependent manner by encapsulating insulin-secreting cells in nonimmunogenic poly(ethylene glycol) diacrylate (PEGDA) hydrogel microspheres. We evaluated cell viability and insulin secretory characteristics of microencapsulated cells. Glucose stimulation studies verified free diffusion of glucose and insulin through the microspheres, while no statistical difference in insulin secretion was observed between cells in microspheres and cells in monolayers. Scratch assays demonstrated accelerated keratinocyte migration in vitro when treated with microencapsulated cells. In excisional wounds on the dorsa of diabetic mice, microencapsulated RIN-m cells accelerated wound closure by postoperative day 7; a statistically significant increase over AtT-20ins-treated and control groups. Histological results indicated significantly greater epidermal thickness in both microencapsulated RIN-m and AtT-20ins-treated wounds. The results suggest that microencapsulation enables insulin-secreting cells to persist long enough at the wound site for a therapeutic effect and thereby functions as an effective delivery vehicle to accelerate wound healing.

Mammalian cells have been microencapsulated within both natural and synthetic polymers for over half a century. Specifically, in the last 36 years microencapsulated cells have been used therapeutically to deliver a wide range of drugs, cytokines, growth factors, and hormones while enjoying the immunoisolation provided by the encapsulating material. In addition to preventing immune attack, microencapsulation prevents migration of entrapped cells. Cells can be microencapsulated in a variety of geometries, the most common being solid microspheres and hollow microcapsules. The micrometer scale permits delivery by injection and is within diffusion limits that allow the cells to provide the necessary factors that are missing at a target site, while also permitting the exchange of nutrients and waste products. The majority of cell microencapsulation is performed with alginate/poly-L-lysine microspheres. Since alginate itself can be immunogenic, for cell-based therapy applications various groups are investigating synthetic polymers to microencapsulate cells. We describe a protocol for the formation of microspheres and microcapsules using the synthetic polymer poly(ethylene glycol) diacrylate (PEGDA).

Low-magnitude, high-frequency vibration has stimulated osteogenesis in mesenchymal stem cells when these cells were cultured in certain types of three-dimensional environments. However, results of osteogenesis are conflicting with some reports showing no effect of vibration at all. A large number of vibration studies using three-dimensional scaffolds employ scaffolds derived from natural sources. Since these natural sources potentially have inherent biochemical and microarchitectural cues, we explored the effect of low-magnitude, high-frequency vibration at low, medium, and high accelerations when mesenchymal stem cells were encapsulated in poly(ethylene glycol) diacrylate microspheres. Low and medium accelerations enhanced osteogenesis in mesenchymal stem cells while high accelerations inhibited it. These studies demonstrate that the isolated effect of vibration alone induces osteogenesis.

In situ measurement to determine mammalian cell number in a non-invasive, non-destructive and reagent-free manner is needed to enable continuous cell manufacturing. An analytical method is presented for non-invasive cell counting by conducting multiwavelength spectral analysis of mammalian cells achieving a minimal detectable cell count of 62,500 at 295 nm. Light absorbance was insensitive to culture volume, giving an absolute cell count rather than a concentration. The activation state of cells was also considered. The study was extended to quantification within polymeric microcapsules as an advanced substrate for mammalian cell growth in bioreactor formats and resulted in an offset directly correlating with the absorbance maxima of the polymer. These studies provide feasibility for optical density as a simple end point to indirectly quantify mammalian cell number for continuous monitoring of cell cultures.

Burn injuries affect approximately 11 million people annually, with fatalities amounting up to 180,000. Burn injuries constitute a global health issue associated with high morbidity and mortality. Recent years have seen advancements in regenerative medicine for burn wound healing encompassing stem cells and stem cell-derived products such as exosomes and conditioned media with promising results compared to current treatment approaches. Sources of stem cells used for treatment vary ranging from hair follicle stem cells, embryonic stem cells, umbilical cord stem cells, to mesenchymal stem cells, such as adipose-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, and even stem cells harvested from discarded burn tissue. Stem cells utilize various pathways for wound healing, such as PI3/AKT pathway, WNT-β catenin pathway, TGF-β pathway, Notch and Hedgehog signaling pathway. Due to the paracrine signaling mechanism of stem cells, exosomes and conditioned media derived from stem cells have also been utilized in burn wound therapy. As exosomes and conditioned media are cell-free therapy and contain various biomolecules that facilitate wound healing, they are gaining popularity as an alternative treatment strategy with significant improvement in outcomes. The treatment is provided either as direct injections or embedded in a natural/artificial scaffold. This paper reviews in detail the different sources of stem cells, stem cell-derived products, their efficacy in burn wound repair, associated signaling pathways and modes of delivery for wound healing.

Summary Cellular therapy is enabling new approaches to tackle significant unmet needs in areas such as regenerative medicine and immunotherapy. The pharmacology of cell therapeutics becomes of critical importance to assure that these new drugs work reproducibly and effectively. Cell pharmacology can benefit from adapting principles of classical molecular drug pharmacokinetics (PK) and pharmacodynamics (PD) to quantitatively understand rate‐limiting constraints of cell fate after administration. Future innovations focused on improvements in drug delivery using a PK/PD perspective can aid in designing a cell therapeutic product to overcome any pharmacological barriers for a given disease application. Herein, we present a perspective on the development of an ex vivo mesenchymal stromal therapeutic using a PK/PD framework and also present examples of general cell engineering techniques that implicitly influence the PK/PD curve by genetically modifying cells to regulate their in vivo duration, biodistribution, and activity. stem cells translational medicine 2019;8:874&879

IntroductionWe previously demonstrated that insulin secreting cells (ISCs) accelerate healing of chronic wounds, and it is known that mesenchymal stem cells (MSCs) also accelerate wound healing. Here, we report that the combination of both cell types coencapsulated into a synthetic hydrogel dressing accelerates chronic wound healing 3 × faster than control and 2 × faster than each cell type delivered singly. Specifically, insulin released by ISCs activates the PI3/Akt pathway, which is vital to the function and survival of MSCs. MSCs in turn improve the viability and function of ISCs.Materials and MethodsMSCs and/or rat islet tumor RIN-m cells were encapsulated into polyethylene glycol diacrylate hydrogel sheets and applied to 1 cm2 full thickness excisional wounds on the dorsa of genetically diabetic male mice (BKS.Cg-m +/+Leprdb/J) in accordance with protocols approved by the Rutgers IACUC. Encapsulated cell viability was assessed using a LIVE/DEAD® Viability/Cytotoxicity Kit. Akt phosphorylation, insulin, VEGF, and TGF-β1 secretion were assessed by ELISA. Animals were sacrificed on postoperative days 14 and 28 and wound tissue was collected for histological and western blot analysis.ResultsISC:MSC combination groups had the highest levels of every secreted product and phosphorylated Akt, and closed wounds in 14 days, ISC-only or MSC-only groups closed wounds in 28 days, control groups closed wounds in 40 days. Further, ISC:MSC groups healed without intermediate scab or scar.ConclusionsCombining MSCs with ISCs results in a more robust healing response than singly delivered cells, warranting further investigation of coencapsulation for MSC therapies.