脫髮長久以來一直是個被高度關注的議題，而目前也發展出許多方式來試著解決之像是植髮、藥物治療、雷射治療等等。然而，上述結果仍由於毛囊的損失以及毛囊周邊細胞的不受控而有所限制。當談到毛髮再生，不只有具有毛髮誘導能力的真皮乳突細胞是必須的，以及上皮與間質的交互作用亦同樣重要。此外，由表皮角質細胞與真皮乳突細胞的參與所觸發的上皮與間質交互作用被認為於影響初始毛髮誘導階段中是不可缺少的。但是，當真皮乳突細胞於平面培養時會逐漸失去其特色能力。另外，脂肪幹細胞作為一能夠由皮下脂肪取得的理想細胞來源同時具有類似骨髓幹細胞的多能性與調節毛髮週期的能力。根據我們先前的研究，塗有甲殼素的培養盤具有提供一三維培養環境能使細胞於其培養下能形成細胞球體，而真皮乳突細胞的毛髮誘導能力能藉由此種三維培養方式來形成細胞球體以保持甚至加強。因此，本篇研究的目的在顯示如何藉由一三維共培養系統並適當模擬毛囊結構的方式將真皮乳突細胞、角質細胞與脂肪幹細胞結合創造一多層狀細胞球體進而促進真皮乳突細胞的毛髮誘導能力。其中真皮乳突細胞、角質細胞與脂肪幹細胞皆取自於C57BL/6小鼠。塗有甲殼素的96孔培養盤能夠提供獨立且合適的三維共培養以形成細胞球。為了評估最佳的層狀球體構造的接種順序，我們採用一致的細胞數量分別是每孔4x104顆真皮乳突細胞、2.5x104顆角質細胞與2.5x104顆脂肪幹細胞並且在六天內順序式接種三種細胞於塗有甲殼素的96孔培養盤內以形成不同的多層狀細胞球。從核心到最外層分別有三種，真皮乳突細胞/角質細胞/脂肪幹細胞、真皮乳突細胞/脂肪幹細胞/角質細胞與一混合外層，真皮乳突細胞/脂肪幹細胞加角質細胞。我們表明了此真皮乳突細胞/角質細胞/脂肪幹細胞順序的多層狀細胞球能夠將真皮乳突細胞的毛髮誘導能力表現提升地最為明顯。再者，當我們深入探討類似上皮與間質的角質細胞與真皮乳突細胞的交互作用，我們準備了培養過角質細胞或脂肪幹細胞的條件培養液療法，和由角質細胞或脂肪幹細胞包住真皮乳突細胞的雙層細胞球，作為不同搭配的比較。有趣的是，在雙層細胞球的物理接觸效果下真皮乳突細胞的毛髮誘導能力表現相較於在條件培養液療法下的化學傳遞效果是獲得提升的，說明了真皮乳突細胞與角質細胞的緊密接觸對於誘導真皮乳提細胞的特質是更有幫助的。除此之外，我們透過體內實驗亦發現此真皮乳突細胞/角質細胞/脂肪幹細胞順序的多層狀細胞球對於誘導毛髮再生是更有效且成功的。

It has been attempted to cure hair loss by several different regimens, including hair transplantation, medication and laser treatment. However, the consequent is still limited as the loss of hair follicle (HF) and surrounding cells are not controllable yet. When it comes to HF regeneration, not only the hair-inductive capacities of dermal papilla (DP) are indispensable but the interaction of epithelium-mesenchyme is also necessarily required. Moreover, Epithelium-mesenchyme interaction triggered by the participation of epidermal keratinocytes (KCs) and DP cells is thought to be absolutely necessary when effecting the initial hair-inducing stage. But, DP cells tend to lose its characteristics when being cultured in adherent status. Additionally, adipose-derived stem cell (ASC) is an ideal cell source isolated from intradermal adipose tissue containing multi-potency as bone marrow mesenchymal stem cell and possesses the ability to regulate hair cycles. Based on our previous studies, chitosan-coated dishes are able to provide a 3D culture environment for cell culture to form cell spheres, and the hair-inductive capacity of DP cells could be maintained even enhanced via sphere formation by 3D culture. Therefore, the purpose of this study is to demonstrate how to facilitate the hair-inductive potentialities of DP cells via a 3D co-culture system with KCs and ASCs and create multi-layer cell spheres as an appropriate mimic of HF structure. C57BL/6 mice were used for mice DP cells, mice KCs, and mice ASCs isolations. Chitosan-coated 96 well plates were able to provide a sole and suitable 3D co-culture microenvironment for sphere formation. In order to evaluate the optimal seeding order for layer-by-layer sphere construction, we adopted the same cell density with 4x104 DP cells, 2.5x104 KCs and 2.5x104 ASCs per well and sequentially seeded three different types of cells into chitosan-coated 96 well plates to form diverse multi-layer cell spheres with different orders in 6 days. From the core to the outer layer were DP/KC/ASC sphere, DP/ASC/KC sphere, and a mixed outer layer was DP/ASC+KC sphere. We showed that the expression of hair-inductive DP markers in multi-layer spheres with this DP/KC/ASC arrangement were raised the most significantly. Furthermore, as we delved to explore the interaction between KCs and DP cells like epithelium versus mesenchyme, we prepared the culturing KCs or ASCs conditional treatments (CM), and two-layer spheres with DP cells surrounded by KCs or ASCs, to compare in different arrangements. Interestingly, the expression of hair-inductive DP markers under the physical contact effect of DP/KC sphere was promoted more in comparison with the chemical delivery effect from any CM, demonstrating that the close contact between DP cells and KCs was more beneficial in inducing DP cells features. Besides, We also found that DP/KC/ASC spheres among other heterotypic spheres were more effective and successful in inducing hair regeneration in vivo.

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