使用低血清培養液繼代培養之牙髓幹細胞及牙根尖乳突幹細胞特性分析

Abstract

穩定且合適的幹細胞來源在牙髓組織再生研究扮演關鍵角色，其中以牙髓幹細胞（dental pulp stem cell, DPSC）與牙根尖乳突幹細胞（stem cell from apical papilla, SCAP）應用最廣。然而幹細胞培養多採用高血清（15-20％）培養液（high serum medium），長期可能導致幹細胞失去幹細胞特性、自我分化以及增加潛在惡性轉化風險，使得在牙髓組織再生研究應用的幹細胞多侷限於第3至第8代。後續有學者提出低血清（2％）培養液並添加生長因子（low serum medium with growth factors, LSM-GF）的配方進行DPSC的分離與繼代培養，證實其幹細胞表面標記可維持至第20代以上而沒有顯著改變，讓DPSC細胞可得性增加。然而這類研究多聚焦於DPSC，並無SCAP相關數據，並且僅有幹細胞標記表現結果，缺少多工分化能力的探討，對於LSM-GF用於繼代培養是否能保有幹細胞特性仍有待進一步研究確認。本研究目的是評估以LSM-GF進行繼代培養的DPSC與SCAP之生長行為與多工分化能力，進而探討使用LSM-GF培養液對於維持不同代數的DPSC與SCAP幹細胞特性的影響效應，評估其在牙髓再生研究應用的可行性。

本研究使用本研究團隊先前完成幹細胞標記鑑定並冷凍保存之DPSC與SCAP進行研究，這些幹細胞於初代培養後即使用LSM-GF進行繼代培養，依照細胞繼代數將其分為三個組別：小於第十代（≦P10）、第十三至第十八代（P13~P18）、大於第二十代（≧ P20）。分別進行細胞增殖、生長曲線以及群落形成能力評估以探討DPSC和SCAP的生長行為特性，同時對不同代數的DPSC與SCAP進行成骨、成脂、軟骨分化誘導實驗，了解幹細胞的多工分化能力在繼代培養後是否產生差異。

研究結果顯示，使用LSM-GF所培養的DPSC與SCAP均呈現細長紡錘狀，DPSC有略大於SCAP的趨勢，且兩者在 ≧ P20細胞均有變大的情形。而 ≦P10與P13~P18的生長曲線相似，生長速度隨著繼代培養次數增加有逐漸下降的現象，在≧ P20會有顯著差異；而相同代數下，SCAP增殖速度較DPSC快速，有較短的倍增時間（doubling time）、更多的群體倍增數（population doubling）以及細胞群落（colony）形成。而在分化能力方面，DPSC與SCAP在培養到 ≧ P20皆可被誘導分化形成鈣化、脂肪與軟骨質而保有多工分化的能力。同時SCAP成骨能力優於DPSC，在較短的時間即可出現鈣化沈積，此外兩種細胞皆隨著繼代數增加，均可在較短的誘導時間即出現鈣化沈積，顯現較強的成骨分化能力。DPSC與SCAP在成脂能力方面沒有明顯的差異，兩種細胞皆會隨著誘導時間增加而有更多的油滴形成，但是不同代數之間沒有明顯的差異。DPSC與SCAP在誘導後兩週皆能看到軟骨分化，兩種細胞在不同代數之間沒有顯著差異。

總結來說，在20代的繼代培養下，無論是DPSC與SCAP均可維持其多工分化能力。而DPSC與SCAP在 ≧ P20後，細胞大小與生長速度均有顯著的改變，但在P18代前的差異不大，可做為合適的幹細胞來源。此外與DPSC 相比，SCAP細胞生長速度較快，且生長行為隨繼代數增加的變化較不明顯，繼代後的穩定性較好，更適合用於牙髓再生研究。

Stable and suitable sources of stem cells play a crucial role in pulp regeneration research, with dental pulp stem cells (DPSC) and stem cells from apical papilla (SCAP) being the most widely used. However, isolating and culturing these dental-derived stem cells using high serum medium (15-20%) is suggested, which may cause the loss of stem cell characteristics, self-differentiation and increase the risk of potential malignant transformation after long-term passage. As a result, stem cells used in pulp regeneration are mostly limited to the 3rd to 8th passages. Subsequently, researchers have proposed a formulation using low serum medium (2%) with the addition of growth factors (LSM-GF) for the isolation and subculture of DPSC, which has been shown to maintain their stem cell surface markers for over the 20th passage, thereby increasing the availability of DPSC cells. However, current studies mostly focus on DPSC and lack SCAP-related data. Only the expression of stem cell markers was evaluated, without investigating their multi-differentiation potential. Further research is needed to confirm whether the stem cell characteristics can be maintained using LSM-GF for subculture. The purpose of this study is to evaluate the growth behaviors and multi-differentiation ability of DPSC and SCAP at different passages using LSM-GF for subculture, and to investigate whether their stem cell characteristics are affected after subculture, assessing its feasibility for application in pulp regeneration research.

In this study, DPSC and SCAP previously identified and cryopreserved by our research team were used. These stem cells were subjected to subculture using LSM-GF immediately after reaching 70-80% confluence in primary culture. Based on the cell passages, they were divided into three groups: less than the 10th passage (≦P10), the 13th to 18th passages (P13~P18), and greater than the 20th passage (≧P20). Cell proliferation, growth curves, and colony-forming ability of DPSC and SCAP at different passages were evaluated. Additionally, osteogenic, adipogenic, and chondrogenic induction experiments were conducted on DPSC and SCAP at different passages to investigate their multi-differentiation ability.

The results showed that both DPSC and SCAP subcultured in LSM-GF exhibited a spindle-shaped morphology, with DPSC slightly larger than SCAP. Both DPSC and SCAP showed an increase in cell size after P20. Before P18, both DPSC and SCAP demonstrated similar growth curves, with a gradual decrease in growth rate as cell passages increased. However, the growth rate decreased significantly in both cell types after P20. Among the same passages, SCAP proliferated faster than DPSC, with a shorter doubling time and more population doublings and colony formation. In terms of multi-differentiation potential, DPSC and SCAP at all passages could differentiate into osteogenic, adipogenic, and chondrogenic lineages while maintaining their multi-differentiation ability. SCAP exhibited superior osteogenic ability compared to DPSC, showing calcified deposition in a shorter induction period. Additionally, both cell types showed early calcified deposition in later passages, indicating a stronger osteogenic differentiation potential. DPSC and SCAP showed similar adipogenic and chondrogenic abilities at different passages. Both cell types showed increased formation of lipid droplets with longer induction time.

In summary, both DPSC and SCAP retained their multi-differentiation ability up to the P20 passage. Significant changes in cell size and growth rate were observed after the P20 passage. However, DPSC and SCAP exhibited similar growth behaviors before the P18 passage, indicating that cells before the P18 passage can be considered suitable sources of stem cells. Moreover, SCAP demonstrated a faster cell growth rate and consistent growth behaviors across different passages compared to DPSC, making it more suitable for dental pulp regeneration research.