Development of a smart bioreactor for mammalian adherent cell expansion for application to cell therapy

细胞治疗，特别是干细胞治疗，在治疗许多疾病和修复人体缺陷组织方面显示出巨大的希望。目前的问题是很难直接从病人或捐赠者那里获得足够数量的细胞。迫切需要找到一种方法来生产大量的高质量细胞用于细胞治疗。拟议的研究项目将开发一种智能生物反应器，该反应器基于热响应性聚合物涂层珠，用于培养和扩大（增加细胞数量）贴壁细胞（因为除了血细胞外，大多数细胞需要附着在表面上才能生长）。温度敏感聚合物聚（n -异丙基丙烯酰胺）（PNIPAAm）在37℃时能够支持细胞粘附和生长，但当温度降至32℃以下时，细胞会从聚合物中分离出来，因此可以很容易地收集。这比传统的细胞培养方法有明显的优势，在传统的细胞培养方法中，通过一种酶（如胰蛋白酶）的作用，通过破坏负责细胞粘附的蛋白质来收获粘附细胞。然而，这种酶也会破坏细胞表面的其他关键蛋白质，从而影响细胞的生长和功能。已经成功地将PNIPAAm移植到平面上，细胞能够生长成薄片，用于修复人体心脏组织缺陷。该项目将研究如何将PNIPAAm嫁接到不同类型的小珠表面进行细胞培养。PNIPAAm接枝微珠已被用于分离生物化合物的色谱，但用于培养人类细胞的研究非常有限。在实验室和工业中，在小珠子上培养贴壁细胞是扩增细胞的常规方法，但是在细胞培养结束时需要酶处理来收获细胞。在这个项目中开发的热敏珠将使细胞与珠分离而不需要破坏性的酶处理。在这个项目中，不同类型的珠粒将被移植PNIPAAm，并测试：(1)它们是否对细胞有任何毒性作用，(2)它们是否支持细胞在珠粒上生长，(3)当温度降至32℃以下时细胞是否能与珠粒分离，(4)细胞在珠粒上培养后仍能保持其功能。此外，还将开发一种专门设计和制造的生物反应器，一种容纳细胞和微珠的容器，并为细胞提供必要的营养和氧气。这种所谓的智能生物反应器将用于生产适合细胞治疗的高质量细胞。

Cell therapy, particularly stem cell therapy, shows great promise for curing many diseases and repairing defective tissues in the human body. The problem currently is the difficulty in acquiring sufficient numbers of cells directly from a patient or donor. There is an urgent need to find a way to produce large quantities of high quality cells for cell therapy. The proposed research project will develop a smart bioreactor based on thermoresponsive polymer coated beads for culturing and expanding (increasing in cell number) adherent cells (as most cells, except blood cells, need to attach onto a surface to grow). The temperature sensitive polymer, poly(N-isopropylacrylamide) (PNIPAAm), has been found to be able to support cell adhesion and growth at 37 degree C, but when the temperature drops below 32 degree C, the cells will detach from the polymer and can thus be collected easily. This has distinctive advantages over the conventional cell culture methods in which adherent cells are harvested by destroying the proteins responsible for cell adhesion through the action of an enzyme such as trypsin. However, this enzyme also damages other crucial proteins on cell surfaces, which can affect cell growth and function. Work has been done to graft PNIPAAm to flat surfaces successfully and cells were able to grow into a thin sheet which was used to repair heart tissue defects in the human body.This project will look at how to graft PNIPAAm onto the surfaces of different types of small beads for cell culture. PNIPAAm grafted beads have already been used in chromatography for separating biological compounds, but very limited research has been done on using them for culturing human cells. Culturing adherent cells on small beads is routinely done both in research laboratories and in industry to expand cells, but the enzymatic treatment is needed to harvest cells at the end of cell culture.The thermosensitive beads to be developed in this project will enable separation of cells from beads without the damaging enzymatic treatment. In the project, different types of beads will be grafted with PNIPAAm and tested to see whether: (1) they have any toxic effects on cells, (2) they support cell growth on them, (3) cells can detach from the beads when the temperature drops below 32 degree C, and (4) cells still retain their function after being cultured on the beads. Also, a purpose-designed and fabricated bioreactor, a vessel to accommodate cells and beads and supply necessary nutrients and oxygen to the cells will be developed. This so-named smart bioreactor will be used to generate high quality cells suitable for cell therapy.