Comparative Analysis of Mesenchymal Stem Cell Cultivation in Fetal Calf Serum, Human Serum, and Platelet Lysate in 2D and 3D Systems

In vitro two-dimensional (2D) and three-dimensional (3D) cultivation of mammalian cells requires supplementation with serum. Mesenchymal stem cells (MSCs) are widely used in clinical trials for bioregenerative medicine and in most cases, in vitro expansion and differentiation of these cells are required before application. Optimized expansion and differentiation protocols play a key role in the treatment outcome. 3D cell cultivation systems are more comparable to in vivo conditions and can provide both, more physiological MSC expansion and a better understanding of intercellular and cell-matrix interactions. Xeno-free cultivation conditions minimize risks of immune response after implantation. Human platelet lysate (hPL) appears to be a valuable alternative to widely used fetal calf serum (FCS) since no ethical issues are associated with its harvest, it contains a high concentration of growth factors and cytokines and it can be produced from expired platelet concentrate. In this study, we analyzed and compared proliferation, as well as osteogenic and chondrogenic differentiation of human adipose tissue-derived MSCs (hAD-MSC) using three different supplements: FCS, human serum (HS), and hPL in 2D. Furthermore, online monitoring of osteogenic differentiation under the influence of different supplements was performed in 2D. hPL-cultivated MSCs exhibited a higher proliferation and differentiation rate compared to HS- or FCS-cultivated cells. We demonstrated a fast and successful chondrogenic differentiation in the 2D system with the addition of hPL. Additionally, FCS, HS, and hPL were used to formulate Gelatin-methacryloyl (GelMA) hydrogels in order to evaluate the influence of the different supplements on the cell spreading and proliferation of cells growing in 3D culture. In addition, the hydrogel constructs were cultivated in media supplemented with three different supplements. In comparison to FCS and HS, the addition of hPL to GelMA hydrogels during the encapsulation of hAD-MSCs resulted in enhanced cell spreading and proliferation. This effect was promoted even further by cultivating the hydrogel constructs in hPL-supplemented media

Xeno-Free In Vitro Cultivation and Osteogenic Differentiation of hAD-MSCs on Resorbable 3D Printed RESOMER

The development of alloplastic resorbable materials can revolutionize the field of implantation technology in regenerative medicine. Additional opportunities to colonize the three-dimensionally (3D) printed constructs with the patient’s own cells prior to implantation can improve the regeneration process but requires optimization of cultivation protocols. Human platelet lysate (hPL) has already proven to be a suitable replacement for fetal calf serum (FCS) in 2D and 3D cell cultures. In this study, we investigated the in vitro biocompatibility of the printed RESOMER® Filament LG D1.75 materials as well as the osteogenic differentiation of human mesenchymal stem cells (hMSCs) cultivated on 3D printed constructs under the influence of different medium supplements (FCS, human serum (HS) and hPL). Additionally, the in vitro degradation of the material was studied over six months. We demonstrated that LG D1.75 is biocompatible and has no in vitro cytotoxic effects on hMSCs. Furthermore, hMSCs grown on the constructs could be differentiated into osteoblasts, especially supported by supplementation with hPL. Over six months under physiological in vitro conditions, a distinct degradation was observed, which, however, had no influence on the biocompatibility of the material. Thus, the overall suitability of the material LG D1.75 to produce 3D printed, resorbable bone implants and the promising use of hPL in the xeno-free cultivation of human MSCs on such implants for autologous transplantation have been demonstrated

Pathogen Inactivation of Cellular Blood Products—An Additional Safety Layer in Transfusion Medicine

In line with current microbial risk reduction efforts, pathogen inactivation (PI) technologies for blood components promise to reduce the residual risk of known and emerging infectious agents. The implementation of PI of labile blood components is slowly but steadily increasing. This review discusses the relevance of PI for the field of transfusion medicine and describes the available and emerging PI technologies that can be used to treat cellular blood products such as platelet and red blood cell units. In collaboration with the French medical device manufacturer Macopharma, the German Red Cross Blood Services developed a new UVC light-based PI method for platelet units, which is currently being investigated in clinical trials

MHC Universal Cells Survive in an Allogeneic Environment after Incompatible Transplantation

Cell, tissue, and organ transplants are commonly performed for the treatment of different diseases. However, major histocompatibility complex (MHC) diversity often prevents complete donor-recipient matching, resulting in graft rejection. This study evaluates in a preclinical model the capacity of MHC class I-silenced cells to engraft and grow upon allogeneic transplantation. Short hairpin RNA targeting β2-microglobulin (RN_shβ2m) was delivered into fibroblasts derived from LEW/Ztm (RT1l) (RT1-Al) rats using a lentiviral-based vector. MHC class I (RT1-A-) expressing and -silenced cells were injected subcutaneously in LEW rats (RT1l) and MHC-congenic LEW.1W rats (RT1u), respectively. Cell engraftment and the status of the immune response were monitored for eight weeks after transplantation. In contrast to RT1-A-expressing cells, RT1-A-silenced fibroblasts became engrafted and were still detectable eight weeks after allogeneic transplantation. Plasma levels of proinflammatory cytokines IL-1α, IL-1β, IL-6, TNF-α, and IFN-γ were significantly higher in animals transplanted with RT1-A-expressing cells than in those receiving RT1-A-silenced cells. Furthermore, alloantigen-specific T-cell proliferation rates derived from rats receiving RT1-A-expressing cells were higher than those in rats transplanted with RT1-A-silenced cells. These data suggest that silencing MHC class I expression might overcome the histocompatibility barrier, potentially opening up new avenues in the field of cell transplantation and regenerative medicine

Gelatin-Methacryloyl (GelMA) Formulated with Human Platelet Lysate Supports Mesenchymal Stem Cell Proliferation and Differentiation and Enhances the Hydrogel’s Mechanical Properties

Three-dimensional (3D) cell culture is a major focus of current research, since cultivation under physiological conditions provides more reliable information about in vivo cell behavior. 3D cell cultures are used in basic research to better understand intercellular and cell-matrix interactions. Moreover, 3D cell culture plays an increasingly important role in the in vitro testing of bioactive substances and tissue engineering. Gelatin-methacryloyl (GelMA) hydrogels of different degrees of functionalization (DoFs) are a versatile tool for 3D cell culture and related applications such as bioprinting. Human platelet lysate (hPL) has already demonstrated positive effects on 2D cell cultures of different cell types and has proven a valuable alternative to fetal calf serum (FCS). Traditionally, all hydrogels are formulated using buffers. In this study, we supplemented GelMA hydrogels of different DoF with hPL during adipose tissue-derived mesenchymal stem cell (AD-MSCs) encapsulation. We studied the effect of hPL supplementation on the spreading, proliferation, and osteogenic differentiation of AD-MSCs. In addition, the influence of hPL on hydrogel properties was also investigated. We demonstrate that the addition of hPL enhanced AD-MSC spreading, proliferation, and osteogenic differentiation in a concentration-dependent manner. Moreover, the addition of hPL also increased GelMA viscosity and stiffness

Generation of HLA-deficient platelets from hematopoietic progenitor cells: GENERATION OF HLA-DEFICIENT PLTs

BACKGROUND: Exposure to allogeneic blood products often leads to the development of human leukocyte antigen (HLA) antibodies. Refractoriness to platelet (PLT) transfusion caused by alloimmunization against HLA Class I antigens constitutes a significant clinical problem. STUDY DESIGN AND METHODS: We developed an RNA interference (RNAi)-based approach to silence the expression of HLA Class I molecules on PLTs derived from CD34+ progenitor cells. A lentiviral-based system was used to express short-hairpin RNA (shRNA) targeting b2-microglobulin (b2m) transcripts in CD34+ progenitor cells. Differentiation to PLTs was performed by incubating progenitor cells in the presence of thrombopoietin and interleukin-3. RESULTS: The transduction of RNAi cassettes containing the sequences for shRNAs targeting b2m caused up to 85% reduction of progenitor cells HLA Class I antigen expression, which was maintained in the culture-derived PLTs. The HLA-deficient PLTs derived from HLA-silenced CD34+ cells proved to be fully functional in in vitro tests when compared to peripheral blood–derived PLTs. CONCLUSIONS: Our data show that in vitro generating HLA Class I–deficient PLTs from hematopoietic progenitor cells prove to be feasible. As malignancy risks associated with insertional mutagenesis are not to be expected in anucleated PLTs, provision of HLA-deficient PLTs from large-scale production units may become reality in the management of patients suffering from PLT transfusion refractoriness