Cryopreservation of human adipose tissues
Cryopreservation of human adipose tissues
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DOI：
10.1016/j.cryobiol.2007.08.012
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发表时间：
2007-12-01
期刊：
影响因子：
2.7
通讯作者：
Pu, L. L. Q.
中科院分区：
文献类型：
--
作者：
Cui, X. D.;Gao, D. Y.;Pu, L. L. Q.
关于脂肪组织冷冻保存的科学研究很少进行。本研究的目的是在体外和体内进行，以开发一种新的冷冻保存方法，可以成功地用于长期保存人类脂肪组织，为未来的临床应用。在这项研究中，从36名成年白色女性患者吸脂后获得脂肪抽吸物样品，并在离心后从中间层收集。在体外研究中，我们从初步实验中选择了合适的冷冻保护剂（CPA）及其浓度和可能的组合。选择二甲亚砜（Me 2SO）和海藻糖的组合作为CPA，最佳浓度为0.5 M Me 2SO和0.2 M海藻糖，然后在整个研究中使用。此外，在冷冻保存后，使用缓慢冷却而不接种（1-2 ℃/min至-30 ℃，然后骤降至-196 ℃储存）和快速升温（在40 ℃水浴中，平均35 ℃/min）实现脂肪组织的最大恢复。通过综合脂肪细胞计数和组织学评价新鲜脂肪抽吸物（第1组）、不含CPA的冷冻保存脂肪抽吸物（第2组）或含CPA的冷冻保存脂肪抽吸物（第3组）。在体内研究中，将新鲜脂肪抽吸物（第1组）、不含CPA的冷冻保存脂肪抽吸物（第2组）或含CPA的冷冻保存脂肪抽吸物（第3组）注射到裸鼠中。在4个月时，在每只动物中收获保留的脂肪抽吸物（脂肪移植物），并评估其重量、体积和组织学。在体外研究中，与第2组相比，第3组脂肪抽吸物的累积活脂肪细胞计数显著更高（2.06 +/- 0.54 x 106 mL（-1）vs. 1.07 +/- 0.41 x 10（6）mL（-1），p < 0.0011）。与第1组相比，第3组的综合活脂肪细胞计数仅略低（2.06 ± 0.54 x 106 mL（-1）vs. 2.57 ± 0.56 x 106 mL（-1），p = 0.083）。在组织学上，与第3组相比，第2组的组织收缩更明显。在体内研究中，在所有3组中均观察到不同程度的注射脂肪移植物吸收。然而，第3组注射的脂肪移植物的保留重量和体积显著大于第2组（均p < 0.0001），但保留重量和体积显著小于第3组（重量，p = 0.009178;体积，p = 0.007836）。在组织学上，在第2组中观察到大量组织纤维化，在第3组中观察到脂肪组织保持良好，仅存在少量组织纤维化。本体外和体内研究的结果首次证明，我们优选的冷冻保存方法，即0.5 M Me 2SO和0.2 M海藻糖作为CPA的组合，以及受控的缓慢冷却和快速复温方案，似乎在人脂肪组织的冷冻保存中提供了最大的恢复结果，并且在进一步改进冷冻保存后可能成为真实的选择。在临床环境中的人类脂肪抽吸物。(C)2007年爱思唯尔公司All rights reserved.
Scientific studies on cryopreservation of adipose tissues have seldom been performed. The purpose of our present study is conducted both in vitro and in vivo to develop a novel cryopreservation method that can be used successfully for long-term preservation of human adipose tissues for possible future clinical application. In this study, samples of adipose aspirates were obtained from 36 adult white female patients after liposuction and collected from the middle layer after centrifugation. In the in vitro study, suitable cryoprotectant agents (CPAs) and their concentrations and possible combinations were selected from our preliminary experiment. A combination of dimethyl sulfoxide (Me2SO) and trehalose as CPA with the optimal concentration (0.5 M Me2SO and 0.2 M trehalose) was chosen and then used throughout the study. In addition, maximal recovery of adipose tissues was achieved after cryopreservation using slow cooling without seeding (1-2 degrees C/min to -30 degrees C, followed by plunging to -196 degrees C for storage) and fast warming (in 40 degrees C water bath, averaging 35 degrees C/min). Fresh adipose aspirates (Group 1), cryopreserved adipose aspirates without CPAs (Group 2), or cryopreserved adipose aspirates with CPAs (Group 3) were evaluated by integrated adipocyte counts and histology. In the in vivo study, fresh adipose aspirates (Group 1), cryopreserved adipose aspirates without CPAs (Group 2), or cryopreserved adipose aspirates with CPAs (Group 3) were injected into a nude mouse. The retained adipose aspirates (fat grafts) were harvested in each animal at 4 months and their weight, volume, and histology was assessed. In the in vitro study, significantly higher integrated viable adipocyte count (2.06 +/- 0.54 x 106 mL(-1) vs. 1.07 +/- 0.41 x 10(6) mL(-1), p < 0.0011) of adipose aspirates was found in Group 3 compared with Group 2. Group 3 had only a marginally lower integrated viable adipocyte count compared with Group 1 (2.06 0.54 x 10(6) mL(-1) vs. 2.57 +/- 0.56 x 106 mL(-1), p = 0.083). Histologically, more tissue shrinkage was evident in Group 2 compared with Group 3. In the in vivo study, various degrees of absorption of injected fat grafts were seen in all 3 groups. However, Group 3 had significantly more retained weight and volume of the injected fat grafts than Group 2 (both p < 0.0001) but had significantly less retained weight and volume than Group 3 (weight, p = 0.009178; volume, p = 0.007836). Histologically, a large amount of tissue fibrosis was seen in Group 2, and reasonably well maintained fatty tissue with only a small amount of tissue fibrosis was seen in Group 3. The results from the present in vitro and in vivo studies, for the first time, demonstrate that our preferred cryopreservation method, the combination of 0.5M Me2SO and 0.2 M trehalose as CPA in addition to the controlled slow cooling and fast rewarming protocol, appears to provide the maximum recovered results in cryopreservation of human adipose tissues and may become a real option after further refinements for cryopreservation of human adipose aspirates in a clinical setting. (C) 2007 Elsevier Inc. All rights reserved.