2-Title: Cytoprotective potential of curcumin and resveratrol during freeze-thaw cycle of buffalo bone marrow derived mesenchymal stem cells

Authors: Navjot Kaur, Manjinder Sharma, Milindmitra K Lonare and Digvijay Singh

Source: Ruminant Science (2020)-9(2):215-222.

How to cite this manuscript: Kaur Navjot, Sharma Manjinder, Lonare MK and Singh Digvijay (2020). Cytoprotective potential of curcumin and resveratrol during freeze-thaw cycle of buffalo bone marrow derived mesenchymal stem cells. Ruminant Science 9(2):215-222.


An endeavour has been made to evaluate the effect of curcumin (CUR) and resveratrol (RES) on the cell viability and apoptosis in buffalo bone marrow derived mesenchymal stem cells (MSCs) during the freeze-thaw cycle. MSCs were isolated and cultured in high glucose DMEM supplemented with 15% FBS. MSCs characterized for alkaline phosphatase (AP) activity and stem cell markers (CD73 and OCT4) showed positive expression. Cells were cryopreserved with CUR @ 3.125 µM and RES @ 0.156 µM alone and in combination (3.125 µM+0.156 µM) in a standardized cryopreservative medium for a period of one month. Standardized cryopreservative medium with 7% DMSO and 3% glycerol in high glucose DMEM served as control. Cell viability was significantly increased (p0.05) with the addition of CUR and RES alone as well as in combination than the control group. Apoptotic index, per cent necrotic cells and per cent dead cells were significantly decreased (p0.05) in bio-antioxidant treated groups. In conclusion, the addition of bio-antioxidants in cryopreservation medium suppressed the apoptosis and improved the post-thaw cell recovery during the freeze-thaw cycle.


Abdillah DA, Setyawan EMN, Oh HJ, Ra K, Lee SH, Kim MJ and Lee BC (2019). Iodixanol supplementation during sperm cryopreservation improves protamine level and reduces reactive oxygen species of canine sperm. Journal of Veterinary Science 20:79-86.

Aliakbari F, Gilani MAS, Amidi F, Baazm M, Korouji M, Izadyar F and Abbasi M (2016). Improving the efficacy of cryopreservation of spermatogonia stem cells by antioxidant supplements. Cellular Reprogramming 18(2):87-95.

Aliakbari F, Gilani MAS, Yazdekhasti H, Koruji M, Asgari HR, Baazm M, Izadyar F, Kharrazi NE, Khanezad M and Abbasi M (2017). Effects of antioxidants, catalase and alpha-tocopherol on cell viability and oxidative stress variables in frozen-thawed mice spermatogonial stem cells. Artificial Cells Nanomedicine Biotechnology 45:63-68.

Amidi F, Rashidi Z, KhosravizadehZ, Khodamoradi K, Talebi A, Navid S and Abbasi M (2019). Antioxidant effects of quercetin in freeze-thawing process of mouse spermatogonial stem cells. Asian Pacific Journal of Reproduction 8(1):7-12.

Baskic D, Popovic S, Ristic P and Arsenijevic N (2006). Analysis of cycloheximide-induced apoptosis in human leukocytes: Fluorescence microscopy using annexin V/propidium iodide versus acridine orange/ethidium bromide. Cell Biology International 30:924-932.

Bissoyi A and Pramanik K (2014). Role of the apoptosis pathway in cryopreservation-induced cell death in mesenchymal stem cells derived from umbilical cord blood. Biopreservation and Biobanking 12(4):246-254.

Csaki C, Mobasheri A and Shakibaei M (2009). Synergistic chondroprotective effects of curcumin and resveratrol in human articular chondrocytes: inhibition of IL-1ß-induced NF-κB-mediated inflammation and apoptosis. Arthritis Research and Therapy 11(6):R165-182.

Deng SL, Sun TC, Yu K, Wang ZP, Zhang BL, Zhang Y, Wang XX, Lian ZX and Liu YX (2017). Melatonin reduces oxidative damage and upregulates heat shock protein 90 expression in cryopreserved human semen. Free Radical Biology and Medicine 113:347-354.

Devi P, Sharma M, Singh D, Lonare MK and Udehiya R (2017). Viability and expression pattern of cryopreserved mesenchymal stem cells derived from buffalo bone marrow. Ruminant Science 6(1):7-12.

Doan CC, Truong NH, Vu NB, Nguyen TT, Nguyen HM, Nguyen KG, Do S, Phan NK and Pham PV (2012). Isolation, culture and cryopreservation of human bone marrow derived mesenchymal stem cells. International Journal of Plant, Animal and Environmental Science 2(2):358-367.

Duru NK, Morshedi M, Schuffner A and Oehninger S (2001). Cryopreservation-thawing of fractionated human spermatozoa and plasma membrane translocation of phosphatidylserine. Fertility and Sterility 75:263-268.

Edamura K, Nakano R, Fujimoto K, Teshima K, Asano K and Tanaka S (2014). Effects of cryopreservation on the cell viability, proliferative capacity and neuronal differentiation potential of canine bone marrow stromal cells. Journal of Veterinary Medical Science 76(4):573-577.

Gade NE, Pratheesh MD, Nath A, Dubey PK, Amarpal, Sharma B, Saikumar G and Sharma GT (2012). Molecular and cellular characterization of buffalo bone marrow-derived mesenchymal stem cells. Reproduction in Domestic Animals 48(3):10-15.

Hamid ZA, Hii W, Abdalla BJ, Yen OB, Latif ES, Mohamed J, Rajab NF, Wah CP, Harto MK, Budin SB and Lin L (2014). The role of Hibiscus sabdariffa L. (Rosella) in maintenance of ex vivo murine bone marrow-derived hematopoietic stem cells. Scientific World Journal DOI: 10.1155/2014/258192.

Heng BC, Clement MV and Cao T (2007). Caspase inhibitor Z-VAD-FMK enhances the freeze-thaw survival rate of human embryonic stem cells. Bioscience Reports 27:257-264.

Lee JH, Jung HK, Han YS, Yoon YM, Yun CW, Sun HY and Lee SH (2016). Antioxidant effects of Cirsium setidens extract on oxidative stress in human mesenchymal stem cells. Molecular Medicine Reports 14(4):3777-84.

Liu H, Zhang S, Liu C, Wu J, Wang Y, Yuan L, Du X, Wang R, Marwa P, Zhuang D and Cheng X (2018). Resveratrol ameliorates Microcystin-LR-Induced testis germ cell apoptosis in rats via SIRT1 signaling pathway activation. Toxins 10(6):235-251.

Liu J, Li X, Lin J, Li Y, Wang T, Jiang Q and Chen D (2016). Sarcandra glabra (Caoshanhu) protects mesenchymal stem cells from oxidative stress: A bioevaluation and mechanistic chemistry. BMC Complementary and Alternative Medicine 16(1): 423-33.

Makashovo OE, Babijchuk LO, Zubova OL and Zubov PM (2016). Optimization of cryopreservation technique for human cord blood nucleated cells using combination of cryoprotectant DMSO and antioxidant N-acetyl-L-cysteine. Problems of Cryobiology and Cryomedicine 26(4):295-307.

Martin M, Ortega FP, Vizuete C, Plaza Da’vila GM, Rodriguez MH and Pena FJ (2015). Depletion of intracellular thiols and increased production of 4-hydroxynonenal that occur during cryopreservation of stallion spermatozoa lead to caspase activation, loss of motility and cell death. Biology of Reproduction 93(6):1-11.

Matsumura K, Hayashi F, Nagashima T and Hyon SH (2013). Long-term cryopreservation of human mesenchymal stem cells using carboxylated poly-l-lysine without the addition of proteins or dimethyl sulfoxide. Journal of Biomaterials Science Polymer Edition 24(12):1484-1497.

Ortega FC, Sotillo GY, Varela FE, Gallardo BJM, Muriel A, Gonzalez FL. Tapia JA and Pena FJ (2008). Detection of apoptosis-like changes during the cryopreservation process in equine sperm. Journal of Andrology 29:213-221.

Ozkavukcu S, Erdemli E, Isik A, Oztuna D and Karahuseyinoglu S (2008). Effects of cryopreservation on sperm parameters and ultrastructural morphology of human spermatozoa. Journal of Assisted Reproduction Genetics. 25:403-411.

Paasch U, Sharma RK, Gupta AK, Grunewald S, Mascha EJ, Thomas AJJ, Glander HJ and Agarwal A (2004). Cryopreservation and thawing is associated with varying extent of activation of apoptotic machinery in subsets of ejaculated human spermatozoa. Biology of Reproduction 71:1828-1837.

Paudel K, Kumar S, Meur S and Kumaresan A (2010). Ascorbic acid, catalase and chlorpromazine reduce cryopreservation-induced damages to crossbred bull spermatozoa. Reproduction in Domestic Animals 45:256-262.

Schuffner A, Morshedi M and Oehninger S (2001). Cryopreservation of fractionated, highly motile human spermatozoa: Effect on membrane phosphatidylserine externalization and lipid peroxidation. Human Reproduction 16:2148-2153.

Shabani H, Zandi M, Ofogi H, Sanjabi MR and Pajooh KH (2017). The effect of combining vitamin E and C on the viability improvement of transfected ovine spermatogonial stem cells after cryopreservation and thawing. Turkish Journal of Veterinary and Animal Sciences 41(5):648-655.

Shinde P, Khan N, Melinkeri S, Kale V and Limaye L (2019). Freezing of dendritic cells with trehalose as an additive in the conventional freezing medium results in improved recovery after cryopreservation. Transfusion 59:686-696.

Singh AK, Jha A, Bit A, Kiassov AP, Rizvanov A, Ojha A, Bhoi P, Patra PK, Kumar A and Bissoyi A (2017). Selaginella bryopteris aqueous extract improves stability and function of cryopreserved human mesenchymal stem cells. Oxidative Medicine and Cellular Longevity DOI: 10.1155/2017/8530656.

Stroh C, Cassens U, Samraj AK, Sibrowski W, Schulze Osthoff K and Los M (2002). The role of caspases in cryoinjury: Caspase inhibition strongly improves the recovery of cryopreserved hematopoietic and other cells. FASEB Journal 16:1651-1653.

Tian Y, Li X, Xie H, Wang X, Xie Y, Chen C and Chen D (2018). Protective mechanism of the antioxidant baicalein toward hydroxyl radical-treated bone marrow-derived mesenchymal stem cells. Molecules 23(1):E223.

Wang C, Xiao R, Cao YL and Yin HY (2017). Evaluation of human platelet lysate and dimethyl sulfoxide as cryoprotectants for the cryopreservation of human adipose-derived stem cells. Biochemical and Biophysical Research Communications 491(1):198-203.

Wang N, Wang F, Gao Y, Yin P, Pan C, Liu W and Wang J (2016). Curcumin protects human adipose-derived mesenchymal stem cells against oxidative stress-induced inhibition of osteogenesis. Journal of Pharmacological Sciences 132(3):192-200.

Zhu M, Huang W Q, Qiu W, Zhu F, Xing W, Chen M, An T, Ao L, Xu X and Huang H  (2017). Curcumin protects mesenchymal stem cells against oxidative stress-induced apoptosis via Akt/mTOR/p70S6K pathway. International Journal of Clinical and Experimental Pathology 10(6):6655-64.