Assembly of Size Selective Multicellular Spheroids of Adipose-Derived Stem/Stromal Cells for Use in Regenerative Tissue Engineering: A Methods and Morphologic Study

J. Murray, B. Mynampati, J. Brant, DO AbbySheffield, Marie Crandall, E. Scott
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Abstract

for Abstract As regenerative scaffolds exhibit varying pore sizes, producing adipose-derived stem/stromal cell (ASC) spheroids of selective sizes to populate these pores may be helpful in autologous tissue engineering. Herein we present two protocols for the initial magnetic sorting of ASCs and the subsequent size selective assembly of ASC spheroids; as the critical quality attribute of ASC identity has been shown previously, only spheroid morphology will be studied. Paramagnetic microbeads (Pro-tein G ligand Dynabeads®, Thermo Fisher Scientific) were used to create single-core paramagnetic immunobeads (scPIBs) and dual core paramagnetic immunobeads (dcPIBs). The scPIBs were created by conjugating the paramagnetic microbeads to ASC-selective primary antibodies (mouse antihuman CD 44, CD73, CD90, CD105, BD Biosciences). The dcPIBs were created by initially conjugating the paramagnetic microbeads to secondary antibodies (mouse IgG) and then conjugating the secondary antibodies to the same ASC-selective primary antibodies. The scPIBs and dcPIBs were then admixed within 15 ml of fresh lipoaspirate respectively. The ASC-scPIBs and ASC-dcPIBs were then magnetically precipitated and subsequently cultured in low adherent conditions for five days. Within twenty minutes, scPIBs isolated an average of 1.2 million putative ASCs (8 x 104 cells per ml of lipoaspirate processed) and dcPIBs isolated an average of 1.3 million putative ASCs (8.7 x 104 cells per ml of lipoaspirate processed). Spheroids comprised of ASC-scPIBs were 19.3 µm (average, +/- 5 µm) and spheroids of the ASC-dcPIBs were 216.7 µm (average, +/- 25 µm). ASCs were magnetically precipitated from fresh lipoaspirate in twenty minutes and subsequently underwent self-assembly of small (approximately 20 µm) or large (approximately 220 µm) ASC spheroids over five days. These protocols may be useful in the rapid development of size-selectable ASC spheroids, which may be particularly useful for autologous toxicology, pharmacology, disease modeling, and tissue regeneration.
用于再生组织工程的脂肪来源干细胞/基质细胞的尺寸选择性多细胞球体的组装:一种方法和形态学研究
摘要由于再生支架具有不同的孔径,产生选择性大小的脂肪来源干/基质细胞(ASC)球体来填充这些孔可能有助于自体组织工程。在此,我们提出了两种方案,用于ASC的初始磁性分选和随后ASC球体的尺寸选择性组装;正如ASC同一性的关键质量属性之前已经显示的那样,将只研究球体形态。顺磁性微珠(Pro tein G配体Dynabeads®,Thermo Fisher Scientific)用于制造单核顺磁性免疫珠(scPIB)和双核顺磁性免疫球(dcPIB)。scPIBs是通过将顺磁性微珠与ASC选择性一级抗体(小鼠抗人CD44、CD73、CD90、CD105、BD Biosciences)偶联而产生的。dcPIB是通过最初将顺磁性微珠与二级抗体(小鼠IgG)偶联,然后将二级抗体与相同的ASC选择性一级抗体偶联而产生的。然后将scPIBs和dcPIBs分别混合在15ml新鲜的脂肪抽吸物中。然后将ASC scPIBs和ASC dcPIBs磁性沉淀,随后在低粘附条件下培养5天。在20分钟内,scPIBs平均分离出120万个推定ASCs(每毫升处理的吸脂物8 x 104个细胞),dcPIBs则平均分离出130万个推定ASC(每毫升加工的吸脂器8.7 x 104个单元)。ASC scPIB组成的球体为19.3µm(平均值+/-5µm),ASC dcPIB的球体为216.7µm(均值+/-25µm)。ASCs在20分钟内从新鲜的吸脂器中磁性沉淀,随后在5天内经历小(约20µm)或大(约220µm)ASC球体的自组装。这些方案可能有助于快速开发可选择大小的ASC球体,这可能对自体毒理学、药理学、疾病建模和组织再生特别有用。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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