우리는 DHR(Department of Health Research), ICMR, 인도 정부(DHR-NRI Grant # R.12015/01/2022-HR)에서 AK에 대한 자금 지원을 인정합니다. SR은 인도 정부 ICMR(Grant # 2020-7593/SCR-BMS)로부터 자금을 지원받았으며 PS는 인도 정부 CSIR로부터 펠로우십을 받았습니다. 또한 유세포 분석에 도움을 준 Ms. Sandhya Tokhi와 Ms. Bhupinder Kaur, 그리고 인프라 지원을 제공한 CSIC(Central Sophisticated Instrumentation Core) 및 찬디가르의 PGIMER에게도 감사드립니다.

연구에 설명된 모든 절차는 찬디가르 PGIMER의 연구소 윤리 위원회(IEC# 9195/PG-12 ITRG/2571-72)의 승인을 받았습니다. 모든 세포 배양 관련 실험은 무균 기술에 따라 Class II 생물 안전 작업대(BSC)에서 수행해야 합니다. 치수는 교정을 위해 제3대구치 발치를 받은 3명(F/14, M/14, M/20) 환자의 건강한 치아에서 얻었다. 검체를 채취하기 전에 찬디가르 PGIMER의 윤리 위원회에서 제공한 지침에 따라 환자/보호자로?...

치과용 치수줄기세포의 1차 배양 확립

<ol>
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줄기 세포는 가소성, 견고성, 면역 조절 특성, 파라크린 메커니즘 및 귀환 효율성으로 인해 수많은 질병을 치료할 수 있는 희망을 품고 있습니다. 치과 치수 조직은 뛰어난 가소성과 재생 능력을 갖춘 줄기 세포의 가장 강력하고 가치 있는 공급원으로 간주됩니다. 여기에서는 세포가 펄프 조직 또는 외식물 조각에서 이동하여 형태학적으로 방추체 모양의 섬유아세포와 유사한 균질한 세포 배양으?...

성체 줄기 세포는 가소성, 파라크린 메커니즘 및 면역 조절 특성으로 인해 세포 유도 치료 및 요법을 위한 강력한 치료 도구로 자리 잡았습니다 1,2,3. 줄기 세포 기반 전임상 연구에서 얻은 고무적인 데이터는 연구자들이 벤치에서 병상으로의 번역을 위해 일하도록 영감을 주었습니다. 줄기 세포 치료에 사용되는 줄기 세포의 유형은 성공적인 결과에 중요한 역할을 합니다. 전임상 및 임상 연구에서 중간엽 줄기 세포(MSC)의 가장 널리 보고된 출처는 골수로 남아 있습니다 4,5. 그러나 골수 유래 줄기 세포(BMSC) 사용의 주요 단점은 희귀한 개체군, 매우 침습적인 분리 절차 및 제한된 확장 능력이라는 것입니다. 따라서 MSC의 대체 공급원이 모색되고 있습니다. 이와 관련하여, 치과 조직은 접근성의 용이성, 엄청난 가소성, 높은 재생 잠재력 및 높은 증식 능력을 가지고 있으며, 이제 줄기 세포의 풍부하고 잠재적인 대체 공급원으로 간주되어왔다 6,7,8,9,10.
치과용 치수줄기세포(DPSC)는 2000년 Gronthos에 의해 분리되어 특성화된 최초의 치아 줄기세포였다11. DPSC는 높은 증식률, 상당한 차별화 가능성, 손쉬운 배양을 통한 접근성 용이성, 그리고 가장 중요한 것은 윤리적 우려 없이 버려진 치아에서 얻을 수 있는 능력 때문에 조직 공학 응용 분야에서 주목을 받고 있습니다12. BMSC 및 지방 유래 줄기 세포(ADSC)와 같은 다른 줄기 세포 소스가 갖는 고립 및 부적절한 자가 재생 능력의 한계는 DPSC에 의해 우회된다13. 인간 DPSC는 인간의 기본 치아, 영구치, 사랑니, 박리 낙엽 치아(SHED) 및 치근단 유두에서 얻을 수 있습니다. 더욱이, DPSC는 일반적으로 폐기되는 상부 치아로부터 분리될 수도 있다14. DPSC는 신경능선 관련 마커를 발현하며 in vitro 및 in vivo 모두에서 신경 세포로 분화할 수 있는 잠재력을 가지고 있습니다 15. 신경원성 잠재력 외에도 DPSC는 특정 분화 조건이 주어질 때 골형성, 연골형성, 지방형성, 간형성 및 근형성과 같은 다른 세포계통으로 분화할 수 있다13. 따라서 이러한 다능성 세포는 세포 기반 치료에 대한 큰 잠재력을 가지고 있으며 다양한 조직의 재생에 사용될 수 있습니다. 연구는 또한 각막 재건16, 심근 경색17 복구에서 DPSC의 잠재적 역할과 사지 허혈18, 알츠하이머 19, 파킨슨병 20 및 노화21와 같은 질병에 대한 잠재적 치료 역할을보고했습니다. 따라서, 치아 조직 유래 줄기세포는 치아 재생뿐만 아니라 눈(16), 심장(17), 간(22), 뼈(23) 등과 같은 비치아 장기의 복구 및 재생에도 사용될 수 있다.
펄프 조직에서 MSC 집단을 분리하는 두 가지 특정 방법이 있습니다 - 효소 소화 및 이식 배양24,25. DPSC의 양과 특성에 큰 차이가 없는 1차 배양물의 성공적인 확립은 이 두 가지 방법 모두에 의해 보고되었다26. 이 연구에서는 섬유아세포 오염을 초래할 수 있는 효소 소화에 비해 조혈 세포 및 내피 세포의 오염 없이 DPSC를 생성하기 때문에 이식 방법(27)에 의한 DPSC의 분리에 초점을 맞췄습니다28.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>6 well cell culture plate</td>
			<td>Costar</td>
			<td>3516</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>Alcian blue stain</td>
			<td>EZstain chondrocyte staining kit, HiMedia</td>
			<td>CCK029</td>
			<td></td>
		</tr>
		<tr>
			<td>alizarin red S stain</td>
			<td>Sigma-Aldrich</td>
			<td>TMS-008</td>
			<td>Osteogenic stain</td>
		</tr>
		<tr>
			<td>Antibiotic cocktail</td>
			<td>Himedia</td>
			<td>A002-5X50ML</td>
			<td>To prevent culture contamination</td>
		</tr>
		<tr>
			<td>Ascorbic Acid</td>
			<td>Himedia</td>
			<td>TC094-25G</td>
			<td>Chondrogenic induction</td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>Gibco</td>
			<td>17504044</td>
			<td>For neural induction</td>
		</tr>
		<tr>
			<td>bFGF ( basic Fibroblast Growth Factor)</td>
			<td>Gibco</td>
			<td>PHG0024</td>
			<td>For neural induction</td>
		</tr>
		<tr>
			<td>CD 105</td>
			<td>BD-Pharmingen</td>
			<td>560839</td>
			<td></td>
		</tr>
		<tr>
			<td>CD 35</td>
			<td>Biolegend</td>
			<td>343604</td>
			<td></td>
		</tr>
		<tr>
			<td>CD 45</td>
			<td>Biolegend</td>
			<td>304006</td>
			<td></td>
		</tr>
		<tr>
			<td>CD 73</td>
			<td>Biolegend</td>
			<td>344016</td>
			<td></td>
		</tr>
		<tr>
			<td>CD 90</td>
			<td>Biolegend</td>
			<td>328107</td>
			<td>Characterization</td>
		</tr>
		<tr>
			<td>cetyl pyridinium chloride (CPC)</td>
			<td>Sigma-Aldrich</td>
			<td>1104006</td>
			<td>For Alizarin Red extraction</td>
		</tr>
		<tr>
			<td>Dexamethasone 21-phosphate disodium</td>
			<td>Sigma-Aldrich</td>
			<td>D1159-100MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td>Himedia</td>
			<td>TS1006-5L</td>
			<td>For washing purpose</td>
		</tr>
		<tr>
			<td>EGF (Epidermal Growth Factor)</td>
			<td>Gibco</td>
			<td>PHG0311</td>
			<td>For hepatic and neural&#160; induction</td>
		</tr>
		<tr>
			<td>EVOS LED microscope</td>
			<td>Invitrogen</td>
			<td></td>
			<td>For&#160; fluorescence imaging</td>
		</tr>
		<tr>
			<td>EZ stain Chondrocyte staining kit</td>
			<td>Himedia</td>
			<td>CCK029-1KT</td>
			<td>Chondro stain Kit</td>
		</tr>
		<tr>
			<td>FACS Canto flow cytometer</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>For cell characterization</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>16000044</td>
			<td>For primary culture</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma-Aldrich</td>
			<td>F2442</td>
			<td>For cell culture</td>
		</tr>
		<tr>
			<td>G5 supplement</td>
			<td>Gibco</td>
			<td>17503012</td>
			<td>For neural induction</td>
		</tr>
		<tr>
			<td>HGF( Hepatocyte Growth Factor)</td>
			<td>Sigma-Aldrich</td>
			<td>H1404</td>
			<td>For hepatic Induction</td>
		</tr>
		<tr>
			<td>HLA-DR</td>
			<td>Biolegend</td>
			<td>307605</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Human TGF-&#946;3</td>
			<td>Peprotech</td>
			<td>#100-36E-10U</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenous acid premix</td>
			<td>Sigma-Aldrich</td>
			<td>I3146</td>
			<td>For hepatic Induction</td>
		</tr>
		<tr>
			<td>ITS premix</td>
			<td>Corning</td>
			<td>354350</td>
			<td></td>
		</tr>
		<tr>
			<td>LDL Uptake Assay kit</td>
			<td>Abcam</td>
			<td>ab133127</td>
			<td>For hepatic characterization</td>
		</tr>
		<tr>
			<td>Low glucose DMEM</td>
			<td>Gibco</td>
			<td>11885-084</td>
			<td>For hepatic induction</td>
		</tr>
		<tr>
			<td>MAP2 antibody</td>
			<td>Sigma-Aldrich</td>
			<td>M4403</td>
			<td>For neural characterization</td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>Gibco</td>
			<td>17502048</td>
			<td>For neural induction</td>
		</tr>
		<tr>
			<td>Neural Basal Media</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td>For neural induction</td>
		</tr>
		<tr>
			<td>NFM antibody</td>
			<td>Sigma-Aldrich</td>
			<td>N4142</td>
			<td>For neural characterization</td>
		</tr>
		<tr>
			<td>Nikon Elipse TS100 microscope</td>
			<td>Nikon</td>
			<td></td>
			<td>For&#160; fluorescence imaging</td>
		</tr>
		<tr>
			<td>Oil Red O</td>
			<td>Sigma-Aldrich</td>
			<td>01391-250Ml</td>
			<td>Adipogenic stain</td>
		</tr>
		<tr>
			<td>Oncostatin M</td>
			<td>R&#38;D Systems</td>
			<td>295-OM-010/CF</td>
			<td>For hepatic Induction</td>
		</tr>
		<tr>
			<td>Petridish</td>
			<td>Tarson</td>
			<td>460090-90MM</td>
			<td>For tissue cutting</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Sigma-Aldrich</td>
			<td>15655-100G</td>
			<td>Osteogenic induction</td>
		</tr>
		<tr>
			<td>Propan-2-ol</td>
			<td>Thermo Fisher</td>
			<td>Q13827</td>
			<td>For Oil Red O extraction</td>
		</tr>
		<tr>
			<td>Sodium pyruvate solution</td>
			<td>Sigma life sciences</td>
			<td>S8636-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Sigma-Aldrich</td>
			<td>T4049</td>
			<td>For cell passaging</td>
		</tr>
		<tr>
			<td>Whatman filter paper</td>
			<td>merck</td>
			<td>WHA1001325</td>
			<td>filter paper</td>
		</tr>
		<tr>
			<td>&#945;- Minimum Essential Media (&#945;-MEM)</td>
			<td>Sigma-Aldrich</td>
			<td>M0643-10X 1L</td>
			<td>Media for primary culture</td>
		</tr>
		<tr>
			<td>&#946;-glycerophosphate disodium salt hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>G9422-50G</td>
			<td></td>
		</tr>
	</tbody>
</table>

primary culture of dental pulp stem cells

인간 치과 치수는 발치된 치아에서 수확할 수 있는 탁월한 재생 능력을 갖춘 유망한 다능성 줄기 세포 저장소를 나타냅니다. 치과용 치수줄기세포(DPSC)의 신경능선 유래 외중간엽 기원은 조직 복구 및 재생에 대한 다각적인 이점으로 인해 높은 수준의 가소성을 부여합니다. 성체 줄기 세포를 채취, 유지 및 증식시키는 다양한 실용적인 방법이 재생 의학에 사용하기 위해 연구되고 있습니다. 이 연구에서는 외식물 배양법에 의해 치아 조직으로부터 1차 중간엽 줄기세포 배양을 확립하는 방법을 보여줍니다. 분리된 세포는 방추체 모양이었고 배양 플레이트의 플라스틱 표면에 부착되었습니다. 이러한 줄기세포의 표현형 특성 분석은 CD90, CD73 및 CD105와 같은 MSC에 대한 ISCT(International Society of Cell Therapy)에서 권장하는 세포 표면 마커의 양성 발현을 보여주었습니다. 또한, 조혈(CD45) 및 내피 마커(CD34)의 발현은 무시할 수 있는 수준이며, HLA-DR 마커의 발현은 2% 미만으로 DPSC 배양의 균질성과 순도를 확인했습니다. 우리는 또한 adipogenic, osteogenic 및 chondrogenic 계통에 대한 분화를 기반으로 한 그들의 다능성을 설명했습니다. 우리는 또한 이러한 세포가 해당 자극 매체를 추가하여 간과 신경 세포로 분화하도록 유도했습니다. 이 최적화된 프로토콜은 실험실 또는 전임상 연구에 활용될 수 있는 고도로 확장 가능한 중간엽 줄기 세포 집단의 배양에 도움이 될 것입니다. DPSC 기반 치료를 시행하기 위한 임상 설정에 유사한 프로토콜을 통합할 수 있습니다.

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.

저자 스포트라이트: Advancing Tissue Regeneration and Disease Modeling with Dental Pulp Stem Cells(치과용 펄프 줄기 세포를 사용한 조직 재생 및 질병 모델링 발전)

치과용 치수줄기세포의 1차 배양

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.

여기에서는 연구자가 이식 방법 6,7,8,9,10을 통해 DPSC의 순수 배양을 확립하고 여러 계통으로 유도하여 다운스트림 응용 분야에 대한 배양의 순도를 확립하는 방법을 설명합니다.
우리는 그림 1A와 같이 환자의 세 번째 어금니...

Read this Scientific Article Protocol about Author Spotlight: Advancing Tissue Regeneration and Disease Modelling with Dental Pulp Stem Cells at JoVE.com

이 논문은 외식물 배양 방법을 사용하여 치수 줄기세포의 1차 배양을 확립하고 ICSCRT 지침에 따라 이러한 세포의 특성을 분석하기 위한 단계별 가이드를 제공합니다. 이 프로토콜에 의해 분리된 세포는 추가 응용을 위한 중간엽 줄기 세포로 간주될 수 있습니다.

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

우리는 질병 모델을 위한 세포 기반 및 무세포 응용 분야에서 DPSC의 잠재력을 조사하는 동시에 조직 재생과 관련된 세포 및 분자 메커니즘을 연구하고 있습니다. 또한, 줄기세포 유래 분비 인자가 줄기세포의 운명을 조절하고 조직 재생을 촉진하는 데 어떤 역할을 하는지 연구하고 있습니다. 현재 우리의 초점은 3D 배양 기술을 사용하여 조직 재생과 관련된 분자 메커니즘, 유전 및 후성 유전 학적 변화에 대한 더 깊은 이해를 얻는 것입니다.당사는 차세대 염기서열분석(next-gen sequencing), RNA 염기서열분석(RNA sequencing), 칩 염기서열분석(chip sequencing)과 같은 고급 기술을 통합하여 다양한 세포 과정을 지배하는 조절 영역을 밝히고 있습니다. DPSC를 분리하기 위해 explant 방법을 사용하면 내피 세포 및 주위 세포와 같은 다른 세포로부터 분리된 균질한 줄기 세포 집단을 효율적으로 수확할 수 있습니다. 이러한 세포 유형은 효소 절차를 사용하여 DPSC를 확립할 때 세포 배양에 남아 있습니다.DPSC를 약물 스크리닝 및 조직 재생을 위한 세포 모델로 사용하는 당사의 연구는 새로운 치료법, 첨단 맞춤형 의학을 개발할 수 있으며, 두개안면 재건 및 치과 발치 후 생체 내 뼈 재생을 포함한 재생 의학에 광범위한 영향을 미칠 수 있습니다. 우리는 현재 DPSC를 사용하여 긴 뼈 및 종골 결함과 같은 뼈 결함을 해결할 수 있는 방법을 연구하고 있습니다. 또한 신경 퇴행 및 녹내장을 포함한 다양한 질병 모델에서 DPSC에서 파생된 분비 분자의 잠재적인 치료 의미를 탐색하고 있습니다.

치과용 치수줄기세포의 1차 배양

Abstract