Cowpea isoflavones enhance the osteoblast differentiation and antioxidant capacity in synergy with vitamin D and ß-carotene: A mechanistic in vitro study.

Background: Osteoporosis (OS) is a pathological condition that makes bones susceptible to fractures by affecting the balance between bone formation and resorption. Recent literature uncovered the possible potential of bioactive compounds with antioxidant mechanisms to counter the issue. Cowpea (CP) isoflavones based on our previous study, vitamin D and natural antioxidant ß-carotene for its pleotropic protective effects were assessed alone and in combination. Aim: The study aims to assess the antioxidant and osteoblast differentiation abilities of cowpea isoflavones alone and in combination of vitamin D (VD) and ß-carotene (BC) in the human osteosarcoma cell line Saos2. Methods: Saos2 cells were maintained in cell culture conditions and concentrations of CP extract (genistein + daidzein), BC and VD required to increase cell proliferation were estimated using MTT assay. Upon treating cells with the EC50 concentrations, lysates were prepared and levels of alkaline phosphatase (ALP) and osteocalcin were evaluated using ELISA. Oxidative stress parameters and osteoblast differentiation markers were evaluated. Results: CP extract (genistein + daidzein), BC and VD concentrations which enhanced the cell proliferation rate were determined and elevated levels of ALP and osteocalcin upon treatment was observed. Anti-oxidant stress parameters studied showed an increase in cells upon treatment compared to control. Significant alterations in levels of protein involved in osteoblast differentiation are observed upon treatment. Conclusion: Cowpea isoflavones has shown a significant activity against OS by elevating antioxidant parameters and inducing osteoblast differentiation in the present study.

Replacement of serum with ocular fluid for cryopreservation of immature testes.

Cryopreservation of immature testis is a feasible approach for germplasm preservation of male animals. Combinations of dimethyl sulfoxide (DMSO) and foetal bovine serum (FBS) are used for testis cryopreservation. However, an alternative to FBS is needed, because FBS is expensive. Buffalo ocular fluid (BuOF), a slaughter house by-product, could be an economical option. The objective of the present study was to assess whether BuOF can replace FBS for cryopreservation of immature mouse (Mus musculus), rat (Rattus norvegicus), and buffalo (Bubalus bubalis) testes. Results showed that rodent and buffalo testes frozen in DMSO (10% for rodents and 20% for buffalo) with 20% FBS or BuOF had similar numbers of viable and DNA-damaged cells (P > 0.05). The expression of cell proliferation- (PCNA) and apoptosis-specific proteins (Annexin V and BAX/BCL2 ratio) were also comparable in mouse and buffalo testes frozen in DMSO with FBS or BuOF (P > 0.05). Interestingly, rat testis frozen in DMSO with BuOF had lower expression of Annexin V protein than testis frozen in DMSO with FBS (P < 0.05). The percentage of meiotic germ cells (pachytene-stage spermatocytes) in xenografts from testis frozen either in DMSO with BuOF or FBS did not significantly differ in rats or buffalo (P > 0.05). These findings provide evidence that BuOF has potential to replace FBS for cryopreservation of immature rodent and buffalo testis. Further investigation is needed to explore whether BuOF can replace FBS for testis cryopreservation of other species.

Ocular Fluid As a Replacement for Serum in Cell Cryopreservation Media.

Cryostorage is of immense interest in biomedical research, especially for stem cell-based therapies and fertility preservation. Several protocols have been developed for efficient cryopreservation of cells and tissues, and a combination of dimethyl sulfoxide (DMSO) and fetal bovine serum (FBS) is commonly used. However, there is a need for an alternative to FBS because of ethical reasons, high cost, and risk of contamination with blood-borne diseases. The objective of the present study was to examine the possibility of using buffalo (Bubalus bubalis) ocular fluid (BuOF) to replace FBS in cryomedia. Frozen-thawed cells, which were cryopreserved in a cryomedia with BuOF, were assessed for viability, early and late apoptosis, and proliferation. Three cell lines (CHO, HEK, and C18-4), mouse embryonic stem (mES) cells, and primary cells, such as mouse embryonic fibroblast (MEF) cells, human peripheral blood mononuclear cells (hPBMCs), and mouse bone marrow cells (mBMCs), were cryopreserved in cryomedia containing 10% DMSO (D10) with 20% FBS (D10S20) or D10 with 20% BuOF (D10O20). For all three cell lines and mES cells cryopreserved in either D10S20 or D10O20, thawed cells showed no difference in cell viability or cell recovery. Western blot analysis of frozen-thawed-cultured cells revealed that the expression of Annexin V and proliferating cell nuclear antigen (PCNA) proteins, and the ratio of BAX/BCL2 proteins were similar in all three cell lines, mES cells, and hPBMCs cryopreserved in D10S20 and D10O20. However, initial cell viability, cell recovery after culture, and PCNA expression were significantly lower in MEF cells, and the BAX/BCL2 protein ratio was elevated in mBMCs cryopreserved in D10O20. Biochemical and proteomic analysis of BuOF showed the presence of several components that may have roles in imparting the cryoprotective property of BuOF. These results encourage further research to develop an efficient serum-free cryomedia for several cell types using BuOF.