Expired platelet concentrate as a source of human platelet lysate for xenogeneic-free culture of human dermal fibroblasts

Dermal fibroblasts have been used clinically to promote wound healing and to reduce wrinkles. Most of the time, fetal bovine serum (FBS) is used for the expansion of fibroblasts. In addition, chemically defined medium can also be used for fibroblast expansion. Nonetheless, both FBS and chemically defined medium are not ideal to culture cells that will be used clinically as FBS has the risk of pathogen transmission and induction of xenogeneic immune response whilst chemically defined medium is extremely expensive. In this study, we examine the potential of using human platelet lysate (hPL) prepared from expired platelet concentrates to culture human dermal fibroblasts. For the experiments, fibroblasts were cultured with 5 and 10% hPL, with 10% FBS as the control group to compare the cell morphology, viability, growth rate, extracellular matrix gene expression and wound healing. Results showed that fibroblasts cultured with hPL were more elongated and smaller in size. The cell viability was higher than 90% for all groups. Expansion with 10% hPL significantly shorten the population doubling time compared to the 5% hPL and 10% FBS groups. However, fibroblasts cultured with hPL have lower expression of type I collagen, type III collagen and fibronection as well as slower wound closure. In summary, hPL has the potential to be used as a serum substitute for FBS to expand fibroblasts as it significantly increases the cell proliferation. However, further studies are required to determine if the changes in the ECM gene expression and migration of the hPL-expanded fibroblasts will affect the efficacy of the cells in promoting in vivo wound healing

Feasibility of Human Platelet Lysate as an Alternative to Foetal Bovine Serum for In Vitro Expansion of Chondrocytes

Osteoarthritis (OA) is a degenerative joint disease that affects a lot of people worldwide. Current treatment for OA mainly focuses on halting or slowing down the disease progress and to improve the patient’s quality of life and functionality. Autologous chondrocyte implantation (ACI) is a new treatment modality with the potential to promote regeneration of worn cartilage. Traditionally, foetal bovine serum (FBS) is used to expand the chondrocytes. However, the use of FBS is not ideal for the expansion of cells mean for clinical applications as it possesses the risk of animal pathogen transmission and animal protein transfer to host. Human platelet lysate (HPL) appears to be a suitable alternative to FBS as it is rich in biological factors that enhance cell proliferation. Thus far, HPL has been found to be superior in promoting chondrocyte proliferation compared to FBS. However, both HPL and FBS cannot prevent chondrocyte dedifferentiation. Discrepant results have been reported for the maintenance of chondrocyte redifferentiation potential by HPL. These differences are likely due to the diversity in the HPL preparation methods. In the future, more studies on HPL need to be performed to develop a standardized technique which is capable of producing HPL that can maintain the chondrocyte redifferentiation potential reproducibly. This review discusses the in vitro expansion of chondrocytes with FBS and HPL, focusing on its capability to promote the proliferation and maintain the chondrogenic characteristics of chondrocytes

Feasibility of Human Platelet Lysate as an Alternative to Foetal Bovine Serum for In Vitro Expansion of Chondrocytes

Osteoarthritis (OA) is a degenerative joint disease that affects a lot of people worldwide. Current treatment for OA mainly focuses on halting or slowing down the disease progress and to improve the patient’s quality of life and functionality. Autologous chondrocyte implantation (ACI) is a new treatment modality with the potential to promote regeneration of worn cartilage. Traditionally, foetal bovine serum (FBS) is used to expand the chondrocytes. However, the use of FBS is not ideal for the expansion of cells mean for clinical applications as it possesses the risk of animal pathogen transmission and animal protein transfer to host. Human platelet lysate (HPL) appears to be a suitable alternative to FBS as it is rich in biological factors that enhance cell proliferation. Thus far, HPL has been found to be superior in promoting chondrocyte proliferation compared to FBS. However, both HPL and FBS cannot prevent chondrocyte dedifferentiation. Discrepant results have been reported for the maintenance of chondrocyte redifferentiation potential by HPL. These differences are likely due to the diversity in the HPL preparation methods. In the future, more studies on HPL need to be performed to develop a standardized technique which is capable of producing HPL that can maintain the chondrocyte redifferentiation potential reproducibly. This review discusses the in vitro expansion of chondrocytes with FBS and HPL, focusing on its capability to promote the proliferation and maintain the chondrogenic characteristics of chondrocytes

Large-Scale Expansion of Human Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are multipotent stem cells with strong immunosuppressive property that renders them an attractive source of cells for cell therapy. MSCs have been studied in multiple clinical trials to treat liver diseases, peripheral nerve damage, graft-versus-host disease, autoimmune diseases, diabetes mellitus, and cardiovascular damage. Millions to hundred millions of MSCs are required per patient depending on the disease, route of administration, frequency of administration, and patient body weight. Multiple large-scale cell expansion strategies have been described in the literature to fetch the cell quantity required for the therapy. In this review, bioprocessing strategies for large-scale expansion of MSCs were systematically reviewed and discussed. The literature search in Medline and Scopus databases identified 26 articles that met the inclusion criteria and were included in this review. These articles described the large-scale expansion of 7 different sources of MSCs using 4 different bioprocessing strategies, i.e., bioreactor, spinner flask, roller bottle, and multilayered flask. The bioreactor, spinner flask, and multilayered flask were more commonly used to upscale the MSCs compared to the roller bottle. Generally, a higher expansion ratio was achieved with the bioreactor and multilayered flask. Importantly, regardless of the bioprocessing strategies, the expanded MSCs were able to maintain its phenotype and potency. In summary, the bioreactor, spinner flask, roller bottle, and multilayered flask can be used for large-scale expansion of MSCs without compromising the cell quality

Human platelet lysate promotes proliferation but fails to maintain chondrogenic markers of chondrocytes

Traditionally, foetal bovine serum (FBS) is used as a serum supplement for stem cell expansion in vitro. However, it is associated with xenoimmunisation and the transmission of animal pathogens, which may cause harm to stem cell recipients. As a safer alternative, human platelet lysate (HPL) has been introduced for propagating stem cells. Chondrocytes are expanded in vitro for cartilage repair via autologous chondrocyte implantation (ACI). In this study, we compare the efficacy of HPL prepared from expired platelet concentrates with that of FBS for promoting the proliferation and maintenance of the chondrogenic markers of primary human chondrocytes expanded in vitro. Chondrocytes were cultured in F12:Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 5% HPL, 10% HPL and 10% FBS. The cell morphology, viability and growth rate were examined from passage 1 (P1) to P3. RNA was isolated from P3 cells for quantitative polymerase chain reaction (qPCR) to determine the gene expression level of the chondrogenic, dedifferentiation and hypertrophic markers. HPL promoted chondrocyte proliferation without compromising cell viability. In addition, the chondrocytes cultured with HPL were smaller. However, HPL failed to maintain the chondrogenic markers, except SOX 9 (SRY-box transcription factor 9), which was upregulated, but not significantly. Nonetheless, HPL also suppressed the expression of type X collagen (Col X), a chondrocyte hypertrophic marker. In summary, we demonstrate the benefits of HPL supplementation in human chondrocyte culture, where it enhances cell proliferation and suppresses chondrocyte hypertrophy. In the future, HPL can be used for the large-scale expansion of chondrocytes for ACI