establishment of primary culture of dental pulp stem cells (dpscs) from human dental tissue

歯髄幹細胞の初代培養の確立

Adult stem cells have transpired into a powerful therapeutic tool for cell-directed treatments and therapies due to their plasticity, paracrine mechanisms, and immunomodulatory properties1,2,3. The encouraging data from stem cell-based preclinical studies have inspired researchers to work for the bench to-bedside translation. The type of stem cells used for stem cell therapy plays a significant role in successful outcomes. In preclinical and clinical studies, the most widely reported source for mesenchymal stem cells (MSCs) remain bone marrow4,5. However, major drawbacks to&#160;using bone marrow-derived stem cells (BMSCs) include their rare population, highly invasive procedures for isolation, and their limited ability to expand. Therefore, alternative sources of MSCs are being explored. In this regard, dental tissues, with their ease of accessibility, enormous plasticity, high regenerative potential, and high proliferative ability, have now been deemed as a rich and potential alternative source of stem cells6,7,8,9,10.
Dental pulp stem cells (DPSCs) were the first type of dental stem cells to be isolated and characterized by Gronthos in 200011. DPSCs have grabbed the attention for tissue engineering applications because of their high proliferation rate, significant differentiation potential, ease of accessibility with effortless culturing, and, most importantly, their ability to be obtained from a discarded tooth without any ethical concern12.&#160;The limitations posed by other stem cell sources, such as BMSCs and adipose-derived stem cells (ADSCs), in their isolation and inadequate self-renewal capacities are circumvented by DPSCs13. Human DPSCs can be obtained from human primary teeth, permanent teeth, wisdom teeth, exfoliated deciduous teeth (SHEDs), and apical papillae. Moreover, DPSCs can also be isolated from supernumerary teeth, which are generally discarded14. DPSCs express neural crest-associated markers and have the potential to differentiate into neuronal cells both in vitro and in vivo15. In addition to their neurogenic potential, DPSCs can differentiate into other cell lineages, such as osteogenic, chondrogenic, adipogenic, hepatic, and myogenic, when given specific differentiation conditions13. Thus, these multipotent cells hold great potential for cell-based therapy and can be employed for the regeneration of various tissues. Studies have also reported the potential role of DPSCs in the reconstruction of the cornea16, repair of myocardial infarction17, and their potential therapeutic role in diseases like limb ischemia18, Alzheimer&#39;s 19, Parkinson&#39;s 20, and aging21. Therefore, dental tissue-derived stem cells can be used not only for dental regeneration, but also for the repair and regeneration of non-dental organs like eyes16, hearts17, livers22, bones23 etc.
There are two particular methods for the isolation of an MSC population from pulp tissue -&#160;enzymatic digestion and explant culture24,25. Successful establishment of primary cultures without any significant difference in the quantity and properties of DPSCs have been reported by both these methods26. In this study, we have focused on the isolation of DPSCs by the explant method27, since this method generates DPSCs without contamination of hematopoietic and endothelial cells, as compared to enzymatic digestion which can result in fibroblast contamination28.

著者スポットライト:歯髄幹細胞による組織再生と疾患モデリングの進歩

歯髄幹細胞の初代培養

We are investigating the potential of DPSCs in cell-based and cell-free applications for disease models, while studying the cellular and molecular mechanisms involved in tissue regeneration. Additionally, we are exploring the role of stem cell-derived secretory factors in controlling stem cell fate and promoting tissue regeneration. Our current focus is on using 3D culture techniques to gain deeper understanding of molecular mechanisms, genetic and epigenetic changes associated with tissue regeneration.We are incorporating advanced techniques such as next-gen sequencing, RNA sequencing, and chip sequencing to unravel regulatory domains governing various cellular processes. Using explant methods to isolate DPSC, a homogeneous stem cell population can be efficiently harvested, free from other cells like endothelial and pericytes. These cell types remain in the cell culture when DPSCs are established using enzymatic procedures.Our research using DPSCs as a cellular model for drug screening and tissue regeneration can develop novel therapies, advanced personalized medicine, and have far-reaching implications in regenerative medicine including craniofacial reconstruction and in vivo bone regeneration after dental extraction. We are currently investigating how DPSCs can be used to address bone defects, such as long bone and calvarial defects. Additionally, we are exploring the potential therapeutic implications of secretory molecules derived from DPSCs in various disease models, including neurodegeneration and glaucoma.

ヒト歯科組織からの歯髄幹細胞(DPSC)の初代培養法の確立

まず、抜いた歯を滅菌生理食塩水ですすいで、血液を取り除きます。滅菌生理食塩水で灌漑しながら、超硬亀裂バリを使用して歯を縦方向に慎重に2つに切断します。その後、歯とその無傷の歯髄を氷上の滅菌α-MEM培地に入れて細胞および組織培養ラボに輸送します。次に、抽出した歯のサンプルを滅菌培養ペトリ皿に入れ、滅菌PBSで3〜4回十分にすすいでください。鋭利な掘削機と鉗子を使用して、空洞の歯髄組織を慎重に取り除き、滅菌PBSで血液の痕跡を取り除くために徹底的に洗浄します。次に、各部分に1〜2ミリリットルの滅菌PBSを入れて、皿の表面を4つの部分に分割します。歯髄組織サンプルを1つの領域に置き、外科用ブレードを使用して細かく切断します。ティッシュピースをPBSで連続した領域に持ち上げて移します。一方、10%FBSを含む非必須アミノ酸を含むα-MEMを約0.8〜1ミリリットル、および抗生物質カクテルを6ウェルプレートに加えます。そして、メディアを均一に広げるためにそれを動かします。歯髄幹細胞の初代培養開発のさまざまな段階を示しています。播種2日目には、歯科組織からの丸みを帯びた気泡型細胞が観察されました。4日目には、組織から出てくる細胞の乱雑なネットワークが観察されました。しかし、13日目までに、外植片から出てきた細胞の数は増加しました。そして、2週目の終わりまでに、ほとんどの外植片は紡錘形の形態を持つ多くの付着細胞に囲まれました。

establishment-primary-culture-dental-pulp-stem-cells-dpscs-from-human

Research

JoVE Journal

Biology

Establishment of Primary Culture of Dental Pulp Stem Cells (DPSCs) from Human Dental Tissue

122 ビュー.  PGIMER. まず、抜いた歯を滅菌生理食塩水ですすいで、血液を取り除きます。滅菌生理食塩水で灌漑しながら、超硬亀裂バリを使用して歯を縦方向に慎重に2つに切断します。その後、歯とその無傷の歯髄を氷上の滅菌α-MEM培地に入れて細胞および組織培養ラボに輸送します。次に、抽出した歯のサンプルを滅菌培養ペトリ皿に入れ、滅菌PBSで3〜4回十分にすすいでください?...

ビデオ: ヒト歯科組織からの歯髄幹細胞(DPSC)の初代培養法の確立

Primary Culture of Dental Pulp Stem Cells

The human dental pulp represents a promising multipotent stem cell reservoir with pre-eminent regenerative competence that can be harvested from an extracted tooth. The neural crest-derived ecto-mesenchymal origin of dental pulp stem cells (DPSCs) bestows a high degree of plasticity that owes to its multifaceted benefits in tissue repair and regeneration. There are various practical ways of harvesting, maintaining, and proliferating adult stem cells being investigated for their use in regenerative medicine. In this work, we demonstrate the establishment of a primary mesenchymal stem cell culture from dental tissue by the explant culture method. The isolated cells were spindle-shaped and adhered to the plastic surface of the culture plate. The phenotypic characterization of these stem cells showed positive expression of the international society of cell therapy (ISCT)-recommended cell surface markers for MSC, such as CD90, CD73, and CD105. Further, negligible expression of hematopoietic (CD45) and endothelial markers (CD34), and less than 2% expression of HLA-DR markers, confirmed the homogeneity and purity of the DPSC cultures. We further illustrated their multipotency based on differentiation to adipogenic, osteogenic, and chondrogenic lineages. We also induced these cells to differentiate into hepatic-like and neuronal-like cells by adding corresponding stimulation media. This optimized protocol will aid in the cultivation of a highly expandable population of mesenchymal stem cells to be utilized in the laboratory or for preclinical studies. Similar protocols can be incorporated into clinical setups for practicing DPSC-based treatments.

This article provides a stepwise guide to establish a primary culture of dental pulps stem cells using the explant culture method and characterization of these cells based on ICSCRT guidelines. The cells isolated by this protocol can be considered as mesenchymal stem cells for further applications.

The human dental pulp represents a promising multipotent stem cell reservoir with pre-eminent regenerative competence that can be harvested from an ...

生物学

To begin, rinse extracted tooth with a sterile saline solution to remove any blood. Carefully cut the tooth longitudinally into two pieces using a carbide fissure burr while irrigating with sterile saline. Afterward, transport the tooth and its intact pulp in sterile alpha-MEM media on ice to the cell and tissue culture laboratory.Then place the extracted tooth sample in sterile culture Petri dish and thoroughly rinse three to four times with sterile PBS. Using a sharp excavator and forceps, carefully remove the cavity pulp tissue and clean a thoroughly with sterile PBS to remove blood traces. Now divide the dish surface into four parts by putting 1 to 2 milliliters of sterile PBS in each part.Place the pulp tissue sample in one area and cut it into small pieces using a surgical blade. Transfer the tissue pieces by lifting them into successive areas with PBS. Meanwhile, add approximately 0.8 to 1 milliliter of alpha-MEM containing non-essential amino acids with 10%FBS and an antibiotic cocktail in a six-well plate.And move it to spread the media uniformly. Various stages of primary culture development of dental pulp stem cells are shown. Rounded bubble type cells from dental tissue were observed on the second day of seeding.A mess-like network of cells coming out of tissue was observed on the fourth day. However, by the 13th day, the number of cells that emerged out of the explant increased. And by the end of the second week, most of the explants got surrounded by many adherent cells with spindle shape morphology.