人類黑色素細胞在幾丁聚醣上之行為：從單層細胞到立體多細胞球

Abstract

本研究之主要目的是設計以幾丁聚醣為高分子基材之黑色素細胞貼片，以應用於治療白斑之黑色素細胞移植。在研發黑色素細胞貼片的過程中，我們發現人類黑色素細胞在幾丁聚醣上有一新的生長方式，即形成立體多細胞的黑色素細胞球。 我們首先探討黑色素細胞在幾丁聚醣上的生長貼附情形及黑色素細胞球的形成條件。我們發現，黑色素細胞在幾丁聚醣上貼附良好。幾丁聚醣可以維持黑色素細胞的生長、型態及黑色素表現。而影響黑色素細胞球形成的因素中，最重要的是細胞的播灑密度。在超過一定的細胞播灑密度之下，黑色素細胞會逐漸形成多細胞聚集，進而形成黑色素細胞球。黑色素細胞球的形成需要完整的細胞骨架包括肌動蛋白及微管，而血清因子在此過程中並非必要。 以連續照相的方式記錄黑色素細胞在幾丁聚醣上的行為時，我們發現黑色素細胞在幾丁聚醣上有較快的移行速度。細胞密度增加可以促進細胞間的接觸，細胞進而藉由移行而聚集成黑色素細胞球。在加入抗E-cadherin的單株抗體後，黑色素無法形成黑色素細胞球。此外，黑色素細胞在剛形成細胞球時，球內的黑色素細胞仍保有細胞分裂能力。因此要形成多細胞球的條件包括細胞必須能夠在生醫材料上有積極的運動及細胞必須有充分的表面黏著分子。黑色素細胞球內的細胞分裂對於黑色素細胞球增大也有貢獻。 功能上，黑色素細胞球內的細胞，超過百分之九十五都保有細胞活性。黑色素細胞球內的黑色素細胞，仍有黑色素表現。因此黑色素細胞球內的細胞仍具有黑色素細胞的表型。當黑色素細胞球接觸到第一型膠原蛋白後，黑色素細胞球漸漸崩解，細胞又恢復成單層多樹突狀的黑色素細胞。為模擬黑色素細胞在移植時可能遭遇的不良環境，我們將細胞置於缺乏血清及生長因子的條件中測試。一般在此環境中，黑色素細胞活性會快速減低。令人驚訝的是，在血清及生長因子缺乏的不良環境中，黑色素細胞球較單層多樹突黑色素細胞有較佳的存活。因此，以幾丁聚醣培養黑色素細胞球的方式，在移植過程中可以提供黑色素細胞一存活優勢。 我們的研究顯示將黑色素細胞在幾丁聚醣上先培養成黑色素細胞球後，可能可以增進移植成功率。而黑色素細胞在幾丁聚醣上的行為也可當作一模型應用在其他生醫領域，用以研究多細胞自行聚集成立體結構的過程。

Vitiligo is characterized by destruction of melanocytes in the epidermis. In this study, we develop a chitosan-based melanocyte patch transplant for vitiligo treatment. In the development of melanocyte patch, a unique phenomenon of human melanocytes is revealed: formation of multicellular spheroids on chitosan surface. We characterize the attachment and growth of human melanocytes as well as the conditions for melanocyte spheroid formation on chitosan surface. Melanocytes have good attachment to chitosan and chitosan is able to support the growth and phenotype expression of melanocytes. Melanocytes spontaneously aggregate and grow into multicellular spheroids when cells are seeded above a critical seeding density. We further demonstrate that spheroid formation requires intact actin and microtubule networks and serum-derived factors are not essential for this process. By use of time-lapse microscopy, we find that, in comparison with melanocytes on tissue culture polystyrene plates, melanocytes migrate faster on chitosan. With increasing seeding densities which enable frequent intercellular contact, cells translocate and aggregate into spheroids. By disrupting surface E-cadherin with monoclonal antibody, melanocyte spheroid formation is inhibited. Moreover, cells within the spheroids are still proliferative when initial spheroids are formed. Hence, active migration on biomaterials and abundant cell surface adhesion molecules that hold cells together are indispensable for melanocyte spheroid formation. Cell proliferation within the spheroids also contributes to the enlargement of spheroids. Functionally, more than 95% of the cells in the spheroids are viable and the cells within the spheroids produce melanin in the cytoplasm. After being seeded on collagen I coated surface, spheroids disintegrate gradually and melanocytes grow back to a physiological dendritic morphology. To simulate the stringent conditions that melanocytes may encounter during transplantation, cells are tested in a serum and growth factor deprived condition which usually compromises melanocyte viability quickly. Surprisingly, compared with the survival of monolayered melanocytes, cell survival of melanocyte spheroids is much enhanced. Hence, culturing melanocytes into spheroids on chitosan surface can provide melanocytes a survival advantage. Our results suggest that chitosan-based melanocyte spheroid patch may increase the engraftment rates during melanocyte transplantation. In addition, the interaction of melanocytes with chitosan can serve as a model for investigation of the self-assembly process of cells in biomedical researches.