• 대한수의학회지
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• 제58권4호
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• pp.211-217
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• 2018

Mesenchymal stem cells (MSCs) are useful candidates for tissue engineering and cell therapy. Physiological cell environment not only connects cells to each other, but also connects cells to the extracellular matrix that provide mechanical support, thus exposing the entire cell surface and activating signaling pathways. Hydrogel is a polymeric material that swells in water and maintains a distinct 3-dimensional (3D) network structure by cross linking. In this study, we investigated the optimized cellular function for canine adipose tissue-derived MSCs (cAD-MSCs) using hydrogel. We observed that the expression levels of Ki67 and proliferating cell nuclear antigen, which are involved in cell proliferation and stemness, were increased in transwell-hydrogel (3D-TN) compared to the transwell-normal (TN). Also, transforming growth factor-${\beta}1$ and SOX9, which are typical bone morphogenesis-inducing factors, were increased in 3D-TN compared to the TN. Collagen type II alpha 1, which is a chondrocyte-specific marker, was increased in 3D-TN compared to the TN. Osteocalcin, which is a osteocyte-specific marker, was increased in 3D-TN compared to the TN. Collectively, preconditioning cAD-MSCs via 3D culture systems can enhance inherent secretory properties that may improve the potency and efficacy of MSCs-based therapies for bone regeneration process.

• 대한의용생체공학회:의공학회지
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• 제23권2호
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• pp.147-153
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• 2002

본 연구에서는 관상의 Poly(lactic-co-glycolic acid) (PLGA) 담체에 대한 인간의 초자연골과 탄성연골의 형성정도를 살펴보았다. 담체는 PLGA의 분자량에 따라서는 110,000 g/mol과 220,000 g/mol을 비교하였고 내경 유지를 위하여 내경측에는 220,000 g/mol, 외경측에는 110,000 g/mol 의 복합체를 만들거나, 비분해성 고분자 폴리에틸렌 튜브와 110,000 g/mol PLGA의 담체와의 결합도 시도하였다. PLGA 담체들은 주사전자현미경으로 단면 구조를 관찰하였다. 각각의 담체에 20세 미만의 환자들의 비중격에서 채취된 초자연골과 귀에서 채취된 탄성연골에서 분리한 연골세포를 심었다. 분리된 연골세포는 두 번의 계대배양을 거쳐 각각의 PLGA 담체에 심었고 일주일동안 생체 외 환경에서 배양하였다. 각각의 세포와 담체의 복합체를 nude mouse의 배부 좌, 우로 피하조직에 이식하고 8주 뒤 H&E 염색으로 조직 검사를 시행하였다. 110,000 g/mol의 PLGA담체의 연골조직은 잘 형성되어 있었지만 그 내경은 유지되지 못하였다. 반면 220,000 g/mol의 PLGA담체의 연골조직은 내경은 유지하였으나 연골조직이 부분적으로 형성되어 있고 성숙한 연골조직의 양이 많지 않았다. 초자연골 세포에 비교하여 탄성연골 세포가 같은 조건하에서 연골조직을 더 많이 형성한 것으로 나타났다. 관상의 유지를 위하여 220,000 9/mol PLGA 담체를 내경측에 110,000 g/mol PLGA 담체를 외경측으로 한 담체에서는 연골조직 형성이 잘 되지 않았으나 내경측에 폴리에틸렌 튜브를 끼운 110,000 g/mol PLGA 담체에서는 조직 형성과 내경유지가 잘 되었고 원래의 담체와 거의 유사한 형태로 유지되었다. 분화된 연골세포도 조직 소견으로 확인할 수 있었다. 이 1mm 내경의 관상 연골조직은 인공 기관지나 식도 등을 위한 동물 실험과 인공 합성 튜브의 대체 등 앞으로 많은 응용분야가 기대된다.

• Journal of Animal Science and Technology
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• 제64권6호
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• pp.1105-1116
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• 2022

Recently, we reported the robust in vitro three-dimensional (3D) expansion of intestinal organoids derived from adult bovine (> 24 months) samples. The present study aimed to establish an in vitro 3D system for the cultivation of intestinal organoids derived from growing cattle (12 months old) for practical use as a potential alternative to in vivo systems for various purposes. However, very few studies on the functional characterization and 3D expansion of adult stem cells from livestock species compared to those from other species are available. In this study, intestinal crypts, including intestinal stem cells, from the small intestines (ileum and jejunum) of growing cattle were isolated and long-term 3D cultures were successfully established using a scaffold-based method. Furthermore, we generated an apical-out intestinal organoid derived from growing cattle. Interestingly, intestinal organoids derived from the ileum, but not the jejunum, could be expanded without losing the ability to recapitulate crypts, and these organoids specifically expressed several specific markers of intestinal stem cells and the intestinal epithelium. Furthermore, these organoids exhibited key functionality with regard to high permeability for compounds up to 4 kDa in size (e.g., fluorescein isothiocyanate [FITC]-dextran), indicating that apical-out intestinal organoids are better than other models. Collectively, these results indicate the establishment of growing cattle-derived intestinal organoids and subsequent generation of apical-out intestinal organoids. These organoids may be valuable tools and potential alternatives to in vivo systems for examining host-pathogen interactions involving epithelial cells, such as enteric virus infection and nutrient absorption, and may be used for various purposes.

• 대한의용생체공학회:의공학회지
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• 제37권5호
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• pp.186-194
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• 2016

In this study, a co-axial flow induced microfluidic chip to fabricate pure collagen type I microfiber via the control of collagen type I and Na-alginate gelation process. The pure collagen type I microfiber was generated by selective degradation of Ca-alginate from 'Core-Shell' structured hydrogel microfiber. To make 'Core-Shell' structure, collagen type I solution was introduced into core channel and 1.5% Na-alginate solution was injected into side channel in microfluidic chip. To evaluatethe 'Core-Shell' structure, the red and green fluorescence substances were mixed into collagen type I and Na-alginate solution, respectively. The fluorescence substances were uniformly loaded into each fiber, and the different fluorescence images were dependent on their location. By immoblizing EpH4-Ras and C6 cells within collagen type I and Na-alginate solution, we sucessfully demonstrated the co-culture of EpH4-Ras and C6 cells with 'Core-Shell' like hydrogel microfiber for 5 days. Only to produce pure collagen type I hydrogel fiber, tri-sodium citrate solution was used to dissolve the shell-like Ca-alginate hydrogel fiber from 'Core-Shell' structured hydrogel microfiber, which is an excellent advantage when the fiber is employed in three-dimensional scaffold. This novel method could apply various application in tissue engineering and biomedical engineering.

• 한국동물생명공학회지
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• 제39권1호
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• pp.2-11
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• 2024

Background: The small intestine plays a crucial role in animals in maintaining homeostasis as well as a series of physiological events such as nutrient uptake and immune function to improve productivity. Research on intestinal organoids has recently garnered interest, aiming to study various functions of the intestinal epithelium as a potential alternative to an in vivo system. These technologies have created new possibilities and opportunities for substituting animals for testing with an in vitro model. Methods: Here, we report the establishment and characterisation of intestinal organoids derived from jejunum tissues of adult pigs. Intestinal crypts, including intestinal stem cells from the jejunum tissue of adult pigs (10 months old), were sequentially isolated and cultivated over several passages without losing their proliferation and differentiation using the scaffold-based and three-dimensional method, which indicated the recapitulating capacity. Results: Porcine jejunum-derived intestinal organoids showed the specific expression of several genes related to intestinal stem cells and the epithelium. Furthermore, they showed high permeability when exposed to FITC-dextran 4 kDa, representing a barrier function similar to that of in vivo tissues. Collectively, these results demonstrate the efficient cultivation and characteristics of porcine jejunum-derived intestinal organoids. Conclusions: In this study, using a 3D culture system, we successfully established porcine jejunum-derived intestinal organoids. They show potential for various applications, such as for nutrient absorption as an in vitro model of the intestinal epithelium fused with organ-on-a-chip technology to improve productivity in animal biotechnology in future studies.

• Journal of Periodontal and Implant Science
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• 제31권1호
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• pp.1-23
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• 2001

Chitosan is biodegradable natural polymer that has been demonstrated its ability to improve wound healing, and calcium metaphosphate(CMP) is a unique class of phosphate minerals having a polymeric structure. In this study, chitosan/CMP and platelet derived growth factor(PDGF-BB) loaded chitosan/CMP sponges were developed, and the effect of the sponges on bone regeneration and their possibility as scaffolds for bone formation by three-dimensional osteoblast culture were examined. PDGF-BB loaded chitosan/CMP sponges were prepared by freeze-drying of a mixture of chitosan solution and CMP powder, and soaking in a PDGF-BB solution. Fabricated sponge retained its 3-dimensional porous structure with $100-200\;{\mu}m$ pores. The release kinetics of PDGF-BB loaded onto the sponge were measured in vitro with $^{125}I-labeled$ PDGF-BB. In order to examine their possibility as scaffolds for bone formation, fetal rat calvarial osteoblastic cells were isolated, cultured, and seeded into the sponges. The cell-sponge constructs were cultured for 28 days. Cell proliferation, alkaline phosphatase activity were measured at 1, 7, 14 and 28 days, and histologic examination was performed. In order to examine the effect on the healing of bone defect, the sponges were implanted into rat calvarial defects. Rats were sacrificed 2 and 4 weeks after implantation and histologic and histomorphometrical examination were performed. An effective therapeutic concentration of PDGF-BB following a high initial burst release was maintained throughout the examination period. PDGF-BB loaded chitosan/CMP sponges supported the proliferation of seeded osteoblastic cells as well as their differentiation as indicated by high alkaline phosphatase activities. Histologic findings indicated that seeded osteoblastic cells well attached to sponge matrices and proliferated in a multi-layer fashion. In the experiments of implantation in rat calvarial defects, histologic and histomorphometric examination revealed that chitosan/CMP sponge promoted osseous healing as compared to controls. PDGF-BB loaded chitosan/CMP sponge further echanced bone regeneration. These results suggested that PDGF-BB loaded chitosan/CMP sponge was a feasable scaffolding material to grow osteoblast in a three-dimentional structure for transplantation into a site for bone regeneration.

• 생명과학회지
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• 제26권11호
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• pp.1313-1319
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• 2016

암은 비균질적으로 구성된 세포집합체로 간질세포 및 세포 외 기질로 구성된 미세환경과 상호작용에 의해 발병, 전이, 심화되는 복잡한 질병이다. 하지만, 기존의 2차원 배양 세포 기반 플랫폼이 3차원적 생체 환경과 암의 비균질성을 대표하기 힘든 한계를 극복하기 위해 스페로이드 배양 세포를 비롯한 다양한 플랫폼 개발이 활발해지고 있다. 본 연구에서는 특히 감염, 염증 및 식이적 환경성 영향력에 민감한 HCT-8 대장암 세포주를 기반으로 하여 3차원 스페로이드 배양법을 보다 효과적인 방법으로 개선하고, 대장암 스페로이드 세포를 기반으로 암의 비균질적인 특질과 항암내성 연구의 간단하고 개선된 플랫폼을 제시고자 하였다. 3차원 배양법 최적화를 위해 물리적 배양환경 조성과 배양배지 구성에 따른 스페로이드 형태형성을 비교 분석하고 암 줄기세포군의 증가 양상을 확인한 결과, 필수요소로 구성된 제한 배지와 균일한 형태의 비부착성 표면 배양접시에서 배양된 스페로이드가 균일한 형태의 구형을 형성하고 암 줄기세포군이 증가함을 확인하였다. 대장암 스페로이드 세포를 기반으로 대장암 치료제인 5-Fluorouracil (5-FU)에 대한 화학적 감응성 변화를 측정한 결과, 암 줄기세포가 5-FU에 대한 화학적 감수성 저해의 원인이 되며, 최적배양 조건에서 암 줄기세포의 약제 내성의 표현이 증대되었다. 이는 암줄기세포의 항암제 내성에 대한 잠재적 위험성을 내포하는 것으로, 이 방법론은 감염, 염증 및 식이적 요인과 연관된 대장암 스페로이드 세포 기반 항암제 약물반응을 검증하기 위해 효과적이면서 간소한 시험법으로 활용될 수 있을 것이다.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제33권5호
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• pp.385-391
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• 2011

Purpose: The periosteum is a well-known source of osteogenic precursor cells for tissue-engineered bone formation. However, cultured endothelial or endothelial-like cells derived from periosteum have not yet been investigated. This study focused on endothelial-like cell culture from the periosteum. Methods: Periosteal tissues were harvested from the mandible during surgical extraction of lower impacted third molars. The tissues were treated with 0.075% type I collagenase in phosphate-buffered saline (PBS) for 1 hr at $37^{\circ}C$ to release cellular fractions. The collagenase was inactivated with an equal volume of DMEM/10% fetal bovine serum (FBS) and the infranatant was centrifuged for 10 min at 2,400 rpm. The cellular pellet was filtered through a $100{\mu}m$ nylon cell strainer, and the filtered cells were centrifuged for 10 min at 2,400 rpm. The resuspended cells were plated into T25 flasks and cultured in endothelial cell basal medium (EBM)-2. Results: Among the hematopoietic markers, CD146 was more highly expressed than CD31 and CD34. The periosteal-derived cells also expressed CD90 and CD166, mesenchymal stem cell markers. Considering that the expression of CD146 was constant and that the expression of CD90 was lower at passage 5, respectively, the CD146 positive cells in passage 5 were isolated using the magnetic cell sorting (MACS) system. These CD146 sorted, periosteal-derived cells formed tube-like structures on Matrigel. The uptake of acetylated, low-density lipoprotein, labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI-Ac-LDL) was also examined in these cells. Conclusion: These results suggest that the CD146-sorted positive cells can be referred to as periosteal-derived CD146 positive endothelial-like cells. In particular, when a co-culture system with endothelial and osteoblastic cells in a three-dimensional scaffold is used, the use of periosteum as a single cell source would be strongly beneficial for bone tissue engineering.