Kong Yahui, Ebrahimpour Pantea, Liu Yu, Yang Chaoxing, Alonso Laura C
Diabetes Center of Excellence, UMass Medical School.
Diabetes Center of Excellence, UMass Medical School; Department of Medicine, Saint Vincent Hospital.
J Vis Exp. 2018 Jun 29(136):57931. doi: 10.3791/57931.
Experiments using isolated pancreatic islets are important for diabetes research, but islets are expensive and of limited abundance. Islets contain a mixed cell population in a structured architecture that impacts function, and human islets are widely variable in cell type composition. Current frequently used methods to study cultured islets include molecular studies performed on whole islets, lumping disparate islet cell types together, or microscopy or molecular studies on dispersed islet cells, disrupting islet architecture. For in vivo islet studies, paraffin-embedded pancreas sectioning is a powerful technique to assess cell-specific outcomes in the native pancreatic environment. Studying post-culture islets by paraffin sectioning would offer several advantages: detection of multiple outcomes on the same islets (potentially even the exact-same islets, using serial sections), cell-type-specific measurements, and maintaining native islet cell-cell and cell-substratum interactions both during experimental exposure and for analysis. However, existing techniques for embedding isolated islets post-culture are inefficient, time consuming, prone to loss of material, and generally produce sections with inadequate islet numbers to be useful for quantifying outcomes. Clinical pathology laboratory cell block preparation facilities are inaccessible and impractical for basic research laboratories. We have developed an improved, simplified bench-top method that generates sections with robust yield and distribution of islets. Fixed islets are resuspended in warm histological agarose gel and pipetted into a flat disc on a standard glass slide, such that the islets are distributed in a plane. After standard dehydration and embedding, multiple (10+) 4 - 5 µm sections can be cut from the same islet block. Using this method, histological and immunofluorescent analyses can be performed on mouse, rat, and human islets. This is an effective, inexpensive, time-saving approach to assess cell-type-specific, intact-architecture outcomes from cultured islets.
使用分离的胰岛进行实验对糖尿病研究很重要，但胰岛昂贵且数量有限。胰岛包含具有结构化结构的混合细胞群体，这种结构会影响功能，而且人类胰岛的细胞类型组成差异很大。目前研究培养胰岛常用的方法包括对整个胰岛进行分子研究，将不同的胰岛细胞类型归为一类，或者对分散的胰岛细胞进行显微镜或分子研究，这会破坏胰岛结构。对于体内胰岛研究，石蜡包埋胰腺切片是一种强大的技术，可在天然胰腺环境中评估细胞特异性结果。通过石蜡切片研究培养后的胰岛将具有几个优点：在同一胰岛上检测多种结果（甚至可能是完全相同的胰岛，使用连续切片）、细胞类型特异性测量，以及在实验暴露期间和分析过程中保持天然胰岛细胞间和细胞与基质的相互作用。然而，现有的培养后分离胰岛包埋技术效率低下、耗时、容易造成材料损失，并且通常产生的切片中胰岛数量不足，无法用于量化结果。临床病理实验室的细胞块制备设施对于基础研究实验室来说难以获得且不实用。我们开发了一种改进的、简化的台式方法，该方法能产生胰岛产量高且分布良好的切片。固定后的胰岛重悬于温热的组织学琼脂糖凝胶中，然后用移液管移至标准载玻片上的平盘中，使胰岛分布在一个平面上。经过标准脱水和包埋后，可以从同一个胰岛块上切出多个（10个以上）4 - 5微米厚的切片。使用这种方法，可以对小鼠、大鼠和人类胰岛进行组织学和免疫荧光分析。这是一种有效、廉价且省时的方法，可用于评估培养胰岛的细胞类型特异性、完整结构的结果。
