Fortifying the angiogenic efficacy of adipose derived stem cell spheroids using spheroid compaction

임광범,김성원,방석호 한국공업화학회 2021 Journal of Industrial and Engineering Chemistry Vol.93 No.-

Transplantation of stem cell spheroids to ischemic tissues has been suggested as an effective method fortreating ischemic disease, but its therapeutic efficacy varies according to the preparation methods forspheroids. We hypothesized that increasing cell compaction while preparing spheroids could improvethe angiogenic efficacy of spheroids. Optimized human adipose derived stem cell (hADSC) spheroidswere shown to upregulate cell adhesion molecule, and angiogenic paracrine factors expression withdownregulated senescence and apoptotic markers expression. We noted that the low seeding density(1104 cells (10KS)) had a far lower probability of forming spheroids than higher seeding densities(3 104 cells (30KS) and 1105 cells (100KS)). The 30KS spheroids demonstrated higher cell density andexpression levels of the angiogenic paracrine factor expression compared to those of 100 KS spheroids. 30KS spheroids with additional incubation (A30KS) showed significantly increased angiogenic paracrinefactors and cell adhesion molecule expressions with decreased senescence expression compared to thosein 30KS spheroids. Transplanting A30KS spheroids showed significantly enhanced therapeuticangiogenesis in mouse hindlimb ischemia model compared to conventional spheroid transplantation. This study successfully defined a set of novel criteria for spheroid culture, which may contribute to futurestem cell therapies.

Multi-Spheroid-Loaded Human Acellular Dermal Matrix Carrier Preserves Its Spheroid Shape and Improves In Vivo Adipose-Derived Stem Cell Delivery and Engraftment

Kim Jie Hyun,이준용 한국조직공학과 재생의학회 2020 조직공학과 재생의학 Vol.17 No.3

BACKGROUND: Current in vivo adult stem cell delivery presents limited clinical effects due to poor engraftment and survival. To overcome current challenges in cell delivery and promote surgical cell delivery for soft tissue repair, a multispheroid- loaded thin sectioned acellular dermal matrix (tsADM) carrier which preserves loaded spheroids’ three-dimensional (3D) structure, was developed. METHODS: Adipose-derived stem cells (ASCs) were used for spheroid delivery. After generating spheroids in 3D cell culture dishes, spheroid plasticity and survival in-between coverslips were evaluated. Spheroids were loaded onto tsADM, their shape changes were followed up for 14 days, and then imaged. Spheroid adhesion stability to tsADM against shear stress was also evaluated. Finally, cell delivery efficacy was compared with cell-seeded tsADM by in vivo implantation and histological evaluation. RESULTS: Spheroids withstood cyclic compression stress and maintained their 3D shape without fusion after 48 h of culture in-between coverslips. Cell survival improved when spheroids were cultured on tsADM in-between the coverslips. Spheroid-loaded tsADM with coverslips maintained their spheroid outline for 14 days of culture whereas without coverslips, the group lost their outline due to spreading after 4 days in culture. Spheroids loaded onto tsADMs were more stable after six rather than 3 days in culture. Spheroid-loaded tsADMs showed about a 2.96-fold higher ASCs transplantation efficacy than cell-seeded tsADMs after 2 weeks of in vivo transplantation. CONCLUSION: These results indicate that transplantation of spheroid-loaded tsADMs significantly improved cell delivery. These findings suggest that a combined approach with other cells, drugs, and nanoparticles may improve cell delivery and therapeutic efficacy.

수수께끼 같은 석기, 여러면석기

박성진(Seong-Jin PARK) 한국구석기학회 2018 한국구석기학보 Vol.- No.37

여러면석기는 한반도 남부 지역에서 흔하게 출토되는 석기로서, 알아보기는 쉽지만, 어떻게 그리고 무엇 때문에 만들었는지를 설명하기는 까다로운 석기이다. 이 논문은 여러면석기에 관한 연구의 전반부로서, 여러면석기에 관한 국내외 학계의 연구사 및 현황을 국내학계에 보고하기 위해 기획되었다. 지금까지 제기된 여러면석기라는 유형에 관한 형태-기술적 정의들을 살펴보면 크게 세 가지 공통점을 찾을 수 있다. 첫째, 석기의 전체적 모습이 구형체라는 점, 둘째, 석기의 겉면에 으스러진 자국이 많다는 점, 그리고 마지막으로 타격각도가 둔각을 이룬다는 점 등이다. 하지만 이와 같은 특징들은 다른 유형의 석기들에서도 공유되는 속성들이기 때문에 여러면석기를 식별하는데 있어서, 특정 속성에 집착하기 보다는 총체적으로 접근해야 한다. 넓은 의미의 여러면석기는 국제 학계의 관례를 따라, 일반적으로 좁은 의미의 여러면석기, 준석구, 석구 등 세 유형으로 분류될 수 있는데, 유적과 돌감에 따라, 유형들 사이에서 기술적 연속성이 관찰되기도 하고 그렇지 않기도 하다. 한편 여러면석기는 보통 세 단계로 구성되는 일련의 과정을 거치면서 완성된다. 여기서 말하는 세 단계란 밑감 선택(1단계), 여러면석기 성형(2단계) 그리고 석구 제작(3단계) 등이다. 하지만 석구가 의도적으로 제작된 석기가 아니라 단지 수명이 다한 몸돌을 망치 또는 공이처럼 사용한 것일 뿐이라는 시각도 만만치 않다. 최근 여러면석기가 망치 또는 공이라고 주장하는 일부 연구자들은 모루의 역할에 주목하여 새로운 연구 성과를 이끌어냈다. 이 가설은 여러면석기가 홀로 사용되었던 도구가 아니라, 모루와 함께 사용된 석기였을 것으로 본다는 측면에서 기존의 망치 가설과 차이가 있다. 국내 학계에서는 대부분 여러면석기를 의도적으로 제작된 석기로 보든, 아니면 비의도적으로 만들어진 석기로 보든, 모두 여러면석기(좁은 의미), 준석구, 석구를 하나의 과정 속에서 각각의 단계를 지시하는 유물로 이해하고 있는 듯하다. 그러나 (좁은 의미) 여러면석기, 준석구 그리고 석구 간에 기술적 연속성을 찾을 수 없는 유적들도 적지 않으므로 여러면석기와 석구 간의 기술적 연속성 여부는 유적의 맥락을 고려하면서 신중히 판단해야 한다. 여러면석기가 의도적으로 만들어진 것이라고 보는 입장, 즉 여러면석기가 하나의 독립적인석기 유형이라고 인식하는 입장에서도 왜 이 석기를 만들었는지에 대해서는 의견이 다양하다. 이 논문에서는 그동안 국내외 학계에 제출되었던 가설들, 즉 사냥 도구라는 설,놀이 또는 운동 기구라는 설 그리고 주술 행위에 이용된 석기라는 설 등을 차례로 살펴 봤다. 그러나 기능에 관한 가설들을 유물 자체에 대한 유형학적 분석만으로는 결코 검증될 수 없으며 오로지 유물의 맥락, 즉 유적 형성학적 분석, 돌감 분석, 공반관계, 유적의 기능 등과 같은 요소들을 총체적으로 파악해야만 비로소 밝혀질 수 있을 뿐이다. Polyhedron is commonly found in Paleolithic sites in the southern part of the Korean peninsula. Polyhedrons are easy to recognize, but it is difficult to tell how and why they were made for the experts. It was designed to report the history and trends of international researches on the polyhedron to the Korean Paleolithic Society. There are three common points in the definitions of the polyhedron reported until now. First, the whole shape of the polyhedron is spherical shape. Second, there are many battering and pounding marks on the surface of the polyhedron, and lastly, the angle of the striking angle is obtuse. However, since these features are shared with other types of such as cores, hammerstones, it is necessary to approach them in a holistic approach rather than sticking to specific attributes in identifying them. Wide-ranging of the polyhedron is divided into three types, the polyhedron in a narrow sense, the sub-spheroids, the spheroids. But it need to take into account if technical continuation existed enter the types depending on the sites and raw materials. Looking at the production process of the spheroids, it is made through three steps, the first, the selection of raw material and blank, second, the shaping of polyhedron and lastly, battering for the smoothing of its surfaces. However, there is a view that the spheroid is not an intentionally made, but the only result of the exhausted core as a hammer stone. Some researchers who have drawn attention to the role of the anvil, insists that the spheroid would be used pair of the anvil. Whether the polyhedron and the spheroids were intentionally or unintentionally made, many of researchers considers them as the indicators in each steps in a single process. However, since there are many sites where technical continuity can not be found among the polyhedron in a narrow sense, the sub-spheroids, the spheroids, the technical continuity should be judged carefully considering the context of the remains. Even if the researchers insisting the stone maker s intention is reflected in the shape of artefacts, there is no consensus on why this stone was made enter them. In this main text, we have looked at hypotheses that have been submitted to domestic and foreign academia in the past, such as the theory that it would be a tool for hunting, the idea that it was a play or exercise tool, and the hypothesis that it was used in magical activities. It is very difficult to verify these hypotheses archeologically, which can be explained through factors such as the analysis of the raw materials, associations of artefacts, and the function of sites.

Transfer, Imaging, and Analysis Plate for Facile Handling of 384 Hanging Drop 3D Tissue Spheroids

Cavnar, Stephen P.,Salomonsson, Emma,Luker, Kathryn E.,Luker, Gary D.,Takayama, Shuichi SAGE Publications 2014 Journal of laboratory automation Vol.19 No.2

<P>Three-dimensional culture systems bridge the experimental gap between in vivo and in vitro physiology. However, nonstandardized formation and limited downstream adaptability of 3D cultures have hindered mainstream adoption of these systems for biological applications, especially for low- and moderate-throughput assays commonly used in biomedical research. Here we build on our recent development of a 384-well hanging drop plate for spheroid culture to design a complementary spheroid transfer and imaging (TRIM) plate. The low-aspect ratio wells of the TRIM plate facilitated high-fidelity, user-independent, contact-based collection of hanging drop spheroids. Using the TRIM plate, we demonstrated several downstream analyses, including bulk tissue collection for flow cytometry, high-resolution low working-distance immersion imaging, and timely reagent delivery for enzymatic studies. Low working-distance multiphoton imaging revealed a cell type–dependent, macroscopic spheroid structure. Unlike ovarian cancer spheroids, which formed loose, disk-shaped spheroids, human mammary fibroblasts formed tight, spherical, and nutrient-limited spheroids. Beyond the applications we describe here, we expect the hanging drop spheroid plate and complementary TRIM plate to facilitate analyses of spheroids across the spectrum of throughput, particularly for bulk collection of spheroids and high-content imaging.</P>

Evaluation of the shape, viability, stemness and osteogenic differentiation of cell spheroids formed from human gingiva-derived stem cells and osteoprecursor cells

Lee, Sung-Il,Ko, Youngkyung,Park, Jun-Beom D.A. Spandidos 2017 Experimental and therapeutic medicine Vol.13 No.6

<P>The present study was performed to create stem cell spheroids from human gingiva-derived stem cells and osteoprecursor cells and to evaluate the maintenance of the stemness, the viability and osteogenic differentiation of the cell spheroids. Gingiva-derived stem cells were isolated, and a total of 6×10<SUP>5</SUP> stem cells and osteoprecursor cells were seeded into concave micromolds at various ratios. Gingiva-derived stem cells and/or osteoprecursor cells formed spheroids in concave microwells. The spheroids demonstrated a smaller diameter when the number of osteoprecursor cells seeded was lower. The majority of cells in the spheroids were identified to be live cells and the cell spheroids preserved viability throughout the experimental period. The cell spheroids, which contained stem cells, were positive for stem-cell markers. Cell spheroids in concave microwells demonstrated a statistically significant increase in alkaline phosphatase activity as time progressed (P<0.05). A statistically significant difference in phosphatase activity was observed in the stem cell alone group when compared with the osteoprecursor cell group at day 5 (P<0.05). Mineralized extracellular deposits were observed in each group after Alizarin Red S staining. Within the limits of the present study, cell spheroids from gingival cells and osteoprecursor cells maintained shape, viability, stemness and osteogenic differentiation potential.</P>

Combining Diced Cartilage with Chondrocyte Spheroids in GelMA Hydrogel: An Animal Study in Diced Cartilage Grafting Technique

Wang Guanhuier,Jin Mengying,Sun Yimou,An Yang,Zhao Zhenmin 한국조직공학과 재생의학회 2023 조직공학과 재생의학 Vol.20 No.2

BACKGROUND: The phenotype maintenance of diced cartilage is a very important factor to reduce cartilage absorption rate in augmentation rhinoplasty. A novel method which combined diced cartilage with chondrocyte spheroids in gelatin methacrylate (GelMA) hydrogel may have potentially good performance in phenotype maintenance, and is worth exploring. METHODS: The complex grafts formed by loading diced cartilage with chondrocyte spheroids into GelMA hydrogel were used as the experimental group, and the grafts formed of diced cartilage in GelMA were used as the control group. The two groups of grafts were implanted subcutaneously in nude mice. After 1 month and 3 months, the grafts were taken for general observation and histological analysis. The diameter changes of cartilage, the nuclei loss of chondrocyte, and glycosaminoglycan secretion were analyzed. RESULTS: Chondrocyte spheroids with obvious proliferation can be seen in the experimental group. Some diced cartilages had become a whole through the interconnection of chondrocyte spheroids. In addition, the diameter of the chondrocyte spheroids—diced cartilage complex in the experimental group increased significantly, and its nuclei loss rate was less than 1/2 of that in the control group. The maintenance of proteoglycans in diced cartilages in the experimental group was significantly better than that in the control group. CONCLUSION: The combination of diced cartilage with chondrocyte spheroids in GelMA hydrogel can significantly reduce the absorption of cartilage extracellular matrix, enhance phenotype maintenance during subcutaneous ectopic implantation, and can produce inter-chondral connections.

Therapeutic spheroid-forming nanotextured surfaces inspired by desert beetle physosterna cribripes

이미현,이해신 한국공업화학회 2015 한국공업화학회 연구논문 초록집 Vol.2015 No.1

Inspired by distinctive hydrophilic/superhydrophobic hybrid micro-/nano-structured surfaces found in Namib desert beetle, we developed spheroid-forming surface called 'spheroform' on which the size of therapeutic spheroids are precisely controlled, and cell viability in the spheroids is increased compared to existing methods. Also, we found that the cellular spheroids formed on spheroform exhibited superior properties due to the unique quasi-spherical environments. Especially, Glucose sensitivity of islet cell spheroids is increased by 200%, and secretion of vascular endothelial growth factor from mesenchymal stem cell spheroids is increased to 300%.

Stem cell spheroids incorporating fibers coated with adenosine and polydopamine as a modular building blocks for bone tissue engineering

Ahmad, Taufiq,Byun, Hayeon,Lee, Jinkyu,Madhurakat Perikamana, Sajeesh Kumar,Shin, Young Min,Kim, Eun Mi,Shin, Heungsoo Elsevier 2020 Biomaterials Vol.230 No.-

<P><B>Abstract</B></P> <P>Although stem cell spheroids offer great potential as functional building blocks for bottom-up bone tissue engineering, delivery of bioactive signals remain challenging. Here, we engineered adenosine-ligand-modified fiber fragments to create a 3D cell-instructive microenvironment for bone. Briefly, the Poly(ι-lactic acid) (PLLA) nanofiber sheet was partially degraded into fragmented fibers (FFs) through aminolysis and adenosine was stably incorporated via one-step polydopamine coating. The SEM and XPS analysis demonstrated that polydopamine assisted adenosine coating efficiency was significantly increased, which led to high coating efficiency of adenosine and its significant retention. The engineered fibers were then assembled into stable spheroids with human-adipose-derived stem cells (hADSCs). The adenosine in the spheroids effectively stimulated A2bR (1.768 ± 0.08) signaling, which further significantly induced the expression of osteogenic markers such as Runx2 (3.216 ± 0.25), OPN (4.136 ± 0.14), OCN (10.16 ± 0.34), and OSX (2.27 ± 0.11) with improved mineral deposition (1.375 ± 0.05 μg per spheroid). In contrast, the adipogenic differentiation of hADSCs was significantly suppressed within the engineered spheroids. Transplantation of engineered spheroids strongly induced osteogenic differentiation of hADSCs in ectopic subcutaneous tissue. Finally, the bone regeneration was significantly enhanced by implanting AP-FF group (59.97 ± 18.33%) as compared to P-FF (27.96 ± 11.14) and defect only (7.97 ± 3.76%). We propose that stem cell spheroids impregnated with engineered fibers enabling adenosine delivery could be promising building blocks for a bottom-up approach to create large tissues for regeneration of damaged bone.</P>

Hierarchically Assembled Mesenchymal Stem Cell Spheroids Using Biomimicking Nanofilaments and Microstructured Scaffolds for Vascularized Adipose Tissue Engineering

Kim, Taek Gyoung,Park, Suk-Hee,Chung, Hyun Jung,Yang, Dong-Yol,Park, Tae Gwan WILEY-VCH Verlag 2010 Advanced functional materials Vol.20 No.14

<P>Composite multicellular spheroids composed of mesenchymal stem cells (MSCs) and synthetic biodegradable nanofilaments are fabricated. Extracellular-matrix-mimicking nanofilaments, prepared from transverse fragmentation of semicrystalline poly(L-lactic acid) nanofibers and subsequent surface modification with cell adhesive peptides, are used to form composite multicellular spheroids with MSCs by cellular self-assembly. The size of the composite spheroids could be readily controlled with the integrated amount of the nanofilaments. The composite spheroids show enhanced adipogenic potential compared to homotypic spheroids. The resultant spheroids are used as building blocks for 3D biohybrid construction with the assistance of a microstructured scaffold fabricated by a direct polymer melt deposition process. An angiogenic growth factor, basic fibroblast growth factor, is also locally delivered in a sustained fashion from the heparinized scaffold surface for facile neovascularization of adipogenic tissue. The produced multiscaled and multifunctional hybrid MSC construct enable the successful formation of vascularized adipose tissue in vivo.</P> <B>Graphic Abstract</B> <P>A multiscale and multifunctional 3D biohybrid construct is fabricated by the hierarchical assembly of composite multicellular spheroids (composed of extracellular matrix-mimicking nanofilaments and mesenchymal stem cells) and bioactive microstructured scaffolds (surface-loaded with angiogenic growth factor). The resulting hybrid construct enables the successful formation of vascularized adipose tissue in vivo. <img src='wiley_img_2010/1616301X-2010-20-14-ADFM201000458-content.gif' alt='wiley_img_2010/1616301X-2010-20-14-ADFM201000458-content'> </P>

초기태아 뇌조직의 기관형 구상체의 형성에 관한 연구

정희원,한종우,김종수,정천기,전상룡,최길수 대한신경외과학회 1996 Journal of Korean neurosurgical society Vol.25 No.12

The present study investigated whether organotypic spheroids derived from human fetal brain tissue, cultured at agarose-overlay media may serve as an optimal in vitro model for multidisciplinary studies in human neurobiology, particularly in the fields of tumor invasiveness and its biochemical mechanism, using light microscopy. electron microscopy and immunohistochemical staining Eight fetal brain tissues of 8-9 weeks of gestation were minced and explanted into agarose-coated culture wells. After three to five days these human fetal brain tissue fragments emerged as spheroids and could be maintained as organotypic spheroids for up to seven weeks. Light and electron microscopic studies of spheroids demonstrated that most cells were poorly differentiated and there were no definite mature neurons or glial cells after enough culture time, but some cells showed certain evidence suggestive of differentiation to neurons or glial cells immunohistochemical staining for glial fibrillary acidic protein(GFAP) and neuron specific enolase(NSE) demonstrated that NSE-positive cells were oval or spherical cells containing abundant cytoplasm and GFAP-positive cells were fibrillary cytoplasma-containing cells which showed some evidence suggestive of differentiation to glial cells by light microscopy. In future, adding some modifications in culture, this organotypic spheroids derived from the human fetal brain may serve as an optimal in vitro model for neurobiology, especially in the field of studies on tumor invasiveness through co-culture with microtumor spheroids.