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The effective use of human-derived cells that are difficult to freeze, such as parenchymal cells and differentiated cells from stem cells, is crucial. A stable supply of damage-sensitive cells, such as differentiated neuronal cells, neurons, and glial cells can contribute considerably to cell therapy. We developed a serum-free freezing solution that is effective for the cryopreservation of differentiated neuronal cells. The quality of the differentiated and undifferentiated SK-N-SH cells was determined based on cell viability, live-cell recovery rate, and morphology of cultured cells, to assess the efficacy of the freezing solutions. The viability and recovery rate of the differentiated SK-N-SH neuronal cells were reduced by approximately 1.5-folds compared to that of the undifferentiated SK-N-SH cells. The viability and recovery rate of the differentiated SK-N-SH cells were remarkably different between the freezing solutions containing 10% DMSO and that containing 10% glycerol. Cryoprotectants such as fetal bovine serum (FBS), antifreeze proteins (sericin), and sugars (maltose), are essential for protecting against freeze damage in differentiated neuronal cells and parenchymal cells. Serum-free alternatives (sericin and maltose) could increase safety during cell transplantation and regenerative medicine. Considering these, we propose an effective freezing solution for the cryopreservation of neuronal cells., Graphical abstract Image 1, Highlights • The timing of freezing during cell differentiation. • More effective serum-free freezing solution for differentiated neuronal cells. • Improving the quality of damage-sensitive cells, such as differentiated neuronal cells.

Autologous chondrocytes in vitro expanded, are used as tools of regenerative therapies for cartilage injuries. However, inability to maintain the hyaline phenotype both in vitro and post in vivo transplantation, remains one of the major hurdles for long term efficacy under clinical settings. We have reported earlier, hyaline phenotype maintenance of both human and rabbit chondrocytes for a long duration both in vitro when cultured conditions using a Thermo-reversible Gelation Polymer (TGP) scaffold-based methodology and in vivo post-transplantation animal model of cartilage damage. Having intrigued by such encouraging outcome, we in this study, analysed the similar TGP culture environment whether would be able to allow in vitro expansion of severe osteoarthritis affected cartilage tissue from elderly patients and evaluated the cells using lectin microarray characterization for pluripotency. Cartilage tissue were obtained from patients (n = 7; age: 60–85 years) undergoing total knee arthroplasty for severe osteoarthritis. Chondrocytes were isolated and cultured in two groups: i. conventional culture without scaffold (2D) and ii. using a TGP scaffold-based culture (3D) up to 18 weeks. In addition to earlier reported findings such as maintenance of hyaline phenotype having been confirmed in this study as well, surface glycoprotein analysis by lectin microarray demonstrated that the α1-2 Fuc recognition lectin (UEA-1) (marker reported in literature for pluripotent stem cells) was found to be more highly expressed in 3D culture compared to 2D culture and even increased over time in 3D culture. We have developed an environment where osteoarthritis affected chondrocytes from the elderly could be cultured up to 18 weeks in vitro using TGP scaffold which express pluripotent cell associated surface glycoproteins compared to the conventional methodology., Highlights • Good quality chondrocytes were grown from cartilage tissue of elderly with severe osteoarthritis for 18 weeks in vitro. • Inflamed donor chondrocytes could be revived to form normal tissue in a 3D in vitro TGP scaffold environment. • Pluripotent stem cell marker UEA-1 in Lectin microarray was positive in TGP- Polymer scaffold grown cartilage.

Introduction Differentiation of hepatocytes and culture methods have been investigated in dogs as a tool to establish liver transplant and drug metabolism examination systems. However, mass culture techniques for canine hepatocytes (cHep) have not been investigated, and it is necessary to construct a suitable culture system. Recently, a protocol called Bud production has attracted attention, and a mixed culture of human and mouse hepatocytes, stem cells, and artificial blood vessels significantly improved the size and formation ratio of spheroids. The purpose of this study was to investigate and improve the in vitro culture of cHep by mixing canine adipose-derived mesenchymal stem cells (cASCs) and human umbilical vein endothelial cells (HUVECs). Methods Spheroid formation ratio and histological examination were evaluated among four culture methods, including cHep alone, two-mix (cHep + cASCs and cHep + HUVEC), and three-mix (cHep + HUVEC + cASCs), on days 0, 4, and 7. Expression levels of liver-related genes (ALB, AFP, α1-AT, CDH1, CYP2E1, CYP3A12, and TAT) were evaluated by quantitative real-time polymerase chain reaction (RT-PCR). Protein expression of albumin, vimentin, and von Willebrand Factor (vWF) was investigated to confirm the location of the hepatocytes. Results The ratio of spheroid formation was 60.2% in the three-mix culture and was significantly improved compared with cHep alone (5.9%) and two-mix; cHep + cASCs (36.2%) and cHep + HUVEC (26.4%) (P, Graphical abstract Image 1, Highlights • cHep spheroids was co-cultured using canine hepatocytes, mesenchymal stem cells, and human umbilical vein endothelial cells. • The ratio of spheroid formation was improved and expression of liver related genes and protein was maintained. • The three-mix culture could promote large spheroids without impairing liver function in canine hepatocyte.