Background: Physiologically, mesenchymal stem cells (MSCs) require optimal preconditioning in the form of low O₂ tension by 1-3% in bone marrow, 10-15% in adipose tissue, and 2-9% in almost all body tissues. On the other hand, seminiferous tubules in testes require 1-6% of O₂ gradation to perform spermatogenesis. Under these conditions, low O₂ level (hypoxia) is required to reinforce the microenvironment to keep forming quiescent cells. Purpose: This study aims to reveal the differences between in-vitro cultured MSCs under normoxic conditions and hypoxic conditions in cell viability, their pluripotency, and proliferative capability. Method: This study is an exploratory in-vitro laboratory study on Bone Marrow Mesenchymal Stem Cells (BMSC) cultures using hypoxic conditions. A total of 21 culture plates were divided into 3 groups. The data was analyzed using the ANOVA Multivariate test. Results: The highest average of viable cells was found in the P2 group, amounting to 89.29 ± 5.36. On the other hand, the least average of viable cells was found in the P0 group, amounting to 63.67 ± 2.56. Conclusion: The cultures with hypoxic conditions and normoxic preconditions are the best culture conditions since they produce cells that were capable of maintaining the pluripotency while still having a better capability of proliferation and viability, compared to direct hypoxic conditions.

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