Manufacturing porcine islets: culture at 22ýýýýC has no advantage above culture at 37ýýýýC: a gene expression evaluation.
Manufacturing porcine islets: culture at 22ýýýýC has no advantage above culture at 37ýýýýC: a gene expression evaluation.
复制标题
制造猪胰岛：22°C 培养比 37°C 培养没有优势：基因表达评估。
DOI：
10.1111/xen.12048
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发表时间：
2013
影响因子：
3.9
通讯作者：
Papas,KlearchosK
中科院分区：
文献类型：
--
作者：
Mueller,KateR;Martins,KyraV;Murtaugh,MichaelP;Schuurman,Henk-Jan;Papas,KlearchosK
背景胰岛的制造过程包括培养步骤，最初引入该步骤是为了简化准备移植物和移植受体的过程的后勤工作。已经提出，在室温下培养比在37 °C下培养具有优势，部分地通过经由优先消除胰岛内的污染细胞（例如过客白细胞）来降低免疫原性。我们使用从成年猪的胰腺分离的胰岛对此进行了研究。方法从三个供体分离猪胰岛，并在37 ℃下培养1天，然后在三种不同的条件下培养：37 ℃培养6天（条件A）; 22 ℃培养6天（条件B）;或22 ℃培养5天，然后37 ℃培养1天（条件C）。通过DNA测量评估恢复率，通过DNA标准化的耗氧率（OCR/DNA）评估存活率，并通过RT-PCR评估一系列9种淋巴细胞标志物，11种淋巴因子和趋化因子以及14种凋亡和应激标志物的基因表达。培养前的平均OCR/DNA值为129-159 nmol/min·mgDNA，培养条件A、B和C下的平均OCR/DNA值分别为259-291、204-212和207-228 nmol/min·mgDNA。无论培养条件如何，在所有三个系列的淋巴细胞标志物、淋巴因子和趋化因子以及凋亡和应激标志物中检测的基因表达在培养7天后表现出统计学显著的降低。这种降低对于条件A是最显著的：特别地，大多数淋巴细胞标志物显示出>10倍的降低，并且淋巴因子和趋化因子系列中的六种标志物也显示出> 10倍的降低;这些降低与培养期间胰岛内存在的免疫细胞的消除一致。凋亡和应激标志物的减少较少。对于在条件B下的培养，基因表达的减少较少，并且在条件C下的培养导致与在条件A下的那些类似的基因表达水平：这表明在37 °C下24小时足以将基因表达水平从在22 °C下培养的胰岛中的那些重新平衡到在37 °C下培养的胰岛中的那些。结果是一致的制剂从three donors.ConclusionsCulture猪胰岛在37 °C培养提供的好处在22 °C文化方面的OCR/DNA的结果和减少表达的基因编码的淋巴细胞标记物，淋巴因子和趋化因子，标记细胞凋亡和压力。
BackgroundThe manufacturing process of islets includes a culture step which was originally introduced to ease the logistics of procedures in preparing the graft and transplant recipient. It has been suggested that culture at room temperature has an advantage over culture at 37 °C, in part by reducing immunogenicity via preferential elimination of contaminating cells (such as passenger leukocytes) within islets. We investigated this using islets isolated from pancreata of adult pigs.MethodsPorcine islets were isolated from three donors and cultured at 37 °C for 1 day, and then under three different conditions: 37 °C for 6 days (condition A); 22 °C for 6 days (condition B); or 22 °C for 5 days followed by 37 °C for 1 day (condition C). Recovery was assessed by DNA measurement, viability by oxygen consumption rate normalized for DNA (OCR/DNA), and gene expression by RT–PCR for a series of 9 lymphocyte markers, 11 lymphokines and chemokines, and 14 apoptotic and stress markers.ResultsPost‐culture islet recoveries were similar for the three culture conditions. Average OCR/DNA values were 129–159 nmol/min·mgDNA before culture, and 259–291, 204–212, and 207–228 nmol/min·mgDNA, respectively, for culture under conditions A, B, and C, respectively. Irrespective of culture condition, examined gene expression in all three series of lymphocyte markers, lymphokines and chemokines, and apoptotic and stress markers manifested a statistically significant decrease upon culture for 7 days. This decrease was most dramatic for condition A: in particular, most of lymphocyte markers showed a >10‐fold reduction and also six markers in the lymphokine and chemokine series; these reductions are consistent with the elimination of immune cells present within islets during culture. The reduction was less for apoptotic and stress markers. For culture under condition B, the reduction in gene expression was less, and culture under condition C resulted in gene expression levels similar to those under condition A: this indicates that 24 h at 37 °C is sufficient to re‐equilibrate gene expression levels from those in islets cultured at 22 °C to those in islets cultured at 37 °C. Results were consistent among the preparations from the three donors.ConclusionsCulture of porcine islets at 37 °C provides benefits over culture at 22 °C with respect to OCR/DNA outcomes and reduced expression of genes encoding lymphocyte markers, lymphokines and chemokines, and markers for apoptosis and stress.