Notodihardjo Sharon Claudia, Morimoto Naoki, Kakudo Natsuko, Mitsui Toshihito, Le Tien Minh, Tabata Yasuhiko, Kusumoto Kenji
Department of Plastic and Reconstructive Surgery, Kansai Medical University, 2-5-1 Shin-machi, Hirakata City, Osaka, 573-1010, Japan.
Laboratory of Biomaterials, Department of Regeneration Science and Engineering, Institute for Frontier Life and Medical Sciences, Kyoto University, 53 Kawahara-cho Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan.
Regen Ther. 2018 Nov 20;10:1-9. doi: 10.1016/j.reth.2018.10.003. eCollection 2019 Jun.
Human platelet lysate (hPL) part of the growth factor cocktail derived from human platelets, which has been applied as a cell growth supplement. The production process is easier in comparison to platelet-rich plasma; thus, hPL is now considered for use in wound healing therapy. However, methods for preserving hPL for more than several months that maintain its bioactivity must be considered, especially for chronic wound treatment. The present study compared the effects of preservation for 9 months using a refrigerator or deep freezer.
We investigated three preservation conditions. In the C-hPL group, hPL was stored at -80 °C in a deep freezer for 9 months; in the CL-hPL group, hPL was cryopreserved for 9 months at -80 °C in a deep freezer then lyophilized; in the L-hPL group, lyophilized hPL was refrigerated at 4 °C for 9 months. The quantity and quality of growth factors in these three groups were measured by an ELISA and in fibroblast cell cultures. Then, gelatin hydrogel discs were impregnated with hPL and its effects with regard to the promotion of wound healing in mice were evaluated by histologic examinations.
The PDGF-BB concentration in C-hPL, CL-hPL and L-hPL was 18,363 ± 370 pg/ml, 11,325 ± 171 pg/ml, and 12,307 ± 348 pg/ml, respectively; the VEGF concentration was 655 ± 23 pg/ml, 454 ± 27 pg/ml, and 499 ± 23 pg/ml, respectively; and the TGF-β1 concentration was 97,363 ± 5418 pg/ml, 73,198 ± 2442 pg/ml, and 78,034 ± 3885 pg/ml, respectively. In cell culture medium, fibroblast cell cultures were better supported in the hPL groups than in the fetal bovine serum group. In the histologic examination of the wound healing process, no differences were observed among the three preserved hPL groups with regard to epithelialization, or granulation tissue or capillary formation. The wounds in all groups had almost healed by day 14.
The stability of growth factors contained in lyophilized hPL is maintained at 4 °C for up to 9 months. This was a versatile preservation method that can be applied in clinical practice.
人血小板裂解物（hPL）是源自人血小板的生长因子混合物的一部分，已被用作细胞生长补充剂。与富血小板血浆相比，其生产过程更简便；因此，hPL目前被考虑用于伤口愈合治疗。然而，必须考虑能保持其生物活性长达数月以上的hPL保存方法，尤其是用于慢性伤口治疗时。本研究比较了使用冰箱或深冻柜保存9个月的效果。
我们研究了三种保存条件。在C-hPL组中，hPL在深冻柜中于-80°C保存9个月；在CL-hPL组中，hPL先在深冻柜中于-80°C冷冻保存9个月，然后进行冻干；在L-hPL组中，冻干的hPL在4°C冰箱中冷藏9个月。通过酶联免疫吸附测定法（ELISA）和在成纤维细胞培养中测量这三组中生长因子的数量和质量。然后，将明胶水凝胶圆盘用hPL浸渍，并通过组织学检查评估其对小鼠伤口愈合促进作用的效果。
C-hPL、CL-hPL和L-hPL中血小板衍生生长因子BB（PDGF-BB）的浓度分别为18363±370 pg/ml、11325±171 pg/ml和12307±348 pg/ml；血管内皮生长因子（VEGF）的浓度分别为655±23 pg/ml、454±27 pg/ml和499±23 pg/ml；转化生长因子-β1（TGF-β1）的浓度分别为97363±5418 pg/ml、73198±2442 pg/ml和78034±3885 pg/ml。在细胞培养基中，hPL组对成纤维细胞培养的支持作用优于胎牛血清组。在伤口愈合过程的组织学检查中，在三组保存的hPL组之间，在表皮形成、肉芽组织或毛细血管形成方面未观察到差异。所有组的伤口在第14天时几乎均已愈合。
冻干hPL中所含生长因子的稳定性在4°C下可维持长达9个月。这是一种可应用于临床实践的通用保存方法。
