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. 2024 Nov 26:735:150834.
doi: 10.1016/j.bbrc.2024.150834. Epub 2024 Oct 16.

Lysate of bovine adipose-derived stem cells accelerates in-vitro development and increases cryotolerance through reduced content of lipid in the in vitro fertilized embryos

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Lysate of bovine adipose-derived stem cells accelerates in-vitro development and increases cryotolerance through reduced content of lipid in the in vitro fertilized embryos

Noriyoshi Manabe et al. Biochem Biophys Res Commun. .

Abstract

Mesenchymal stem cells such as adipose-derived stem cells (ADSCs) are known to secrete factors that stimulate cell division and promote regeneration in neighboring cells. While conditioned medium from stem cells has been used in blastocyst production, no studies have examined the use of cell lysates. In this study we investigated the effects of adding ADSC lysate to in vitro culture (IVC) medium. ADSCs and fibroblasts were isolated from bovine adipose tissue and auricular tissue, respectively, and their lysates were prepared by freeze-thaw disruption. ADSC lysate was added to synthetic oviductal fluid medium. The effects on cleavage, blastocyst development rates, cell numbers, cryotolerance, gene expression (POU5F1, BAX, IGF1R, IGF2R, SOD2), lipid content, and membrane integrity were evaluated according to the experimental design. In Expt. 1, the comparison involved adding ADSC or fibroblast lysate alongside the control group. The total blastocyst rate increased when ADSC lysate was introduced (ADSCs: 40.1 %, fibroblasts: 33.1 %, control: 27.3 %). However, there were no significant differences in the number of trophoblast cells or in the inner cell mass. Experiment 2 confirmed that this increase in blastocyst development was not due to the solvent, PBS(-). In Expt. 3, addition of 10 % fetal calf serum (FCS) or ADSC lysate increased the total blastocyst rate compared to the control (control, 26.2 %; 10 % FCS, 43.4 %; 1 % ADSC lysate, 34.2 %; 10 % ADSC lysate, 48.1 %). After freezing and thawing, the survival and hatching rates of embryos with FCS were significantly lower than those of the control as well as those with added ADSC lysate. In Expt. 4, the addition of ADSC lysate or FCS had no significant effect on gene expression in blastocysts compared to control. However, the addition of FCS significantly increased the gray intensity, indicating higher lipid content compared to the control, with a significant increase in the number of dead cells in the blastocyst. These results indicate that the addition of ADSC lysate to the IVC medium accelerates bovine blastocyst development and that its 10 % addition, corresponding to 1 × 105 cells/mL, is as effective as 10 % FCS without a decrease in cryotolerance due to the increased lipid content.

Keywords: Adipose-derived stem cell; Blastocyst; Bovine; Cryotolerance; In vitro culture; Lysate.

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Conflict of interest statement

Declaration of competing interest Tokunori Yamamoto is President of Meis Technology, Inc., which possesses a patent related to this work (Japan Patent No. 6970853). The other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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