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.

Fabrication of core-shell spheroids as building blocks for engineering 3D complex vascularized tissue

Kim, Eun Mi,Lee, Yu Bin,Kim, Se-jeong,Park, Jaesung,Lee, Jinkyu,Kim, Sung Won,Park, Hansoo,Shin, Heungsoo Elsevier Science B.V. Amsterdam 2019 ACTA BIOMATERIALIA Vol. No.

<P><B>Abstract</B></P> <P>Cell spheroids as building blocks for engineering micro-tissue should be able to mimic the complex structure of natural tissue. However, control of the distribution of multiple cell populations within cell spheroids is difficult to achieve with current spheroid-harvest methods such as hanging-drop and with the use of microwell plates. In this study, we report the fabrication of core-shell spheroids with the ultimate goal to form 3D complex micro-tissue. We used endothelial cells and two types of stem cells (human turbinate mesenchymal stem cells (hTMSCs)/adipose-derived stem cells (ADSCs)). The stem cells and endothelial cells formed layered micro-sized cell sheets (µCSs) on polydopamine micro-patterned temperature-responsive hydrogel surfaces by a sequential seeding method, and these layered µCSs self-assembled to form core-shell spheroids by expansion of the hydrogels. The co-cultured spheroids formed a core-shell structure irrespective of stem cell type. In addition, the size of the core-shell spheroids was controlled from 90 ± 1 to 144 ± 3 µm by changing pattern sizes (200, 300, and 400 µm). The shell thickness gradually increased from 12 ± 3 to 30 ± 6 µm by adjusting the endothelial cell seeding density. Finally, we fabricated the micro-tissue by fusion of the co-cultured spheroids, and the spheroids with the core-shell structure rapidly induced <I>in vitro</I> vessel-like network in 3 days. Thus, the position of endothelial cells in co-cultured spheroids may be an important factor for the modulation of the vascularization process, which can be useful for the production of 3D complex micro-tissues using spheroids as building blocks.</P> <P><B>Statement of significance</B></P> <P>This manuscript describes our work on the fabrication of core-shell spheroids as building blocks to form 3D complex vascularized micro-tissue. Stem cells (human turbinate mesenchymal stem cells (hTMSCs) or adipose-derived stem cells (ADSCs)) and endothelial cells formed layered micro-sized cell sheets (µCSs) on micro-patterned temperature-responsive hydrogel surfaces by a sequential seeding method, and these layered µCSs self-assembled to form core-shell spheroids (core – stem cells, shell – endothelial cells), irrespective of stem cell type. In addition, the size and shell thickness of the core-shell spheroids were controlled by modifying pattern size and endothelial cell seeding density. We fabricated the vascularized micro-tissue by fusion of the spheroids and demonstrated that the spheroids with a core-shell structure rapidly induced vessel-like network.</P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Two-Cell Spheroid Angiogenesis Assay System Using Both Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Shah, Sajita,Kang, Kyu-Tae The Korean Society of Applied Pharmacology 2018 Biomolecules & Therapeutics(구 응용약물학회지) Vol.26 No.5

Most angiogenesis assays are performed using endothelial cells. However, blood vessels are composed of two cell types: endothelial cells and pericytes. Thus, co-culture of two vascular cells should be employed to evaluate angiogenic properties. Here, we developed an in vitro 3-dimensional angiogenesis assay system using spheroids formed by two human vascular precursors: endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs). ECFCs, MSCs, or ECFCs+MSCs were cultured to form spheroids. Sprout formation from each spheroid was observed for 24 h by real-time cell recorder. Sprout number and length were higher in ECFC+MSC spheroids than ECFC-only spheroids. No sprouts were observed in MSC-only spheroids. Sprout formation by ECFC spheroids was increased by treatment with vascular endothelial growth factor (VEGF) or combination of VEGF and fibroblast growth factor-2 (FGF-2). Interestingly, there was no further increase in sprout formation by ECFC+MSC spheroids in response to VEGF or VEGF+FGF-2, suggesting that MSCs stimulate sprout formation by ECFCs. Immuno-fluorescent labeling technique revealed that MSCs surrounded ECFC-mediated sprout structures. We tested vatalanib, VEGF inhibitor, using ECFC and ECFC+MSC spheroids. Vatalanib significantly inhibited sprout formation in both spheroids. Of note, the $IC_{50}$ of vatalanib in ECFC+MSC spheroids at 24 h was $4.0{\pm}0.40{\mu}M$, which are more correlated with the data of previous animal studies when compared with ECFC spheroids ($0.2{\pm}0.03{\mu}M$). These results suggest that ECFC+MSC spheroids generate physiologically relevant sprout structures composed of two types of vascular cells, and will be an effective pre-clinical in vitro assay model to evaluate pro- or anti-angiogenic property.

Optimization of Tumor Spheroid Preparation and Morphological Analysis for Drug Evaluation

Lee Jaehun,Kim Youngwon,임지석,Jung Hyo-Il,Castellani Gastone,Piccinini Filippo,곽봉섭 한국바이오칩학회 2024 BioChip Journal Vol.18 No.1

Due to its similarity to in vivo conditions, tumor spheroids are actively used in research areas, such as drug screening and cell–cell interactions. A substantial quantity of spheroids is crucial for obtaining dependable results in high-throughput screening. Conventional fabrication methods of spheroid have limitations in low yield and morphological variation. Droplet- based microfluidic system capable of mass-producing uniformed spheroids can overcome these limitations. In this study, we investigated the optimal culture conditions, which allows to researchers provide guidelines for producing spheroids with the desired diameter and quantity. Mass-produced spheroids were employed to analyze compaction, which is crucial for evaluating the remission effects of drugs, as well as the formation of a necrotic core, which induces a bias in the analysis of drug response and viability. The time point at which compaction is completed and the diameter begins to increase was measured using spheroids with diameters of both > 400 μm and < 400 μm, and spheroids do not proliferate a linear growth trend. Spheroid with diameters ranging from 73.4 ± 11.42 μm to 371 ± 5.11 μm was used to predict the formation of the necrotic core based on live cell counting, and diameter of 300–330 μm was mathematically calculated as the diameter where a necrotic core forms. Additionally, the use of artifi cial intelligence (AI) for high-throughput analysis is crucial for obtain- ing time-saving and reproducible data. We produced BT474 and MCF-7 spheroids with diameters of 100, 200, and 300 μm and obtained morphological indicators from an AI-based program to compare the diff erences in heterogeneous breast tumor spheroids. Through this study, we optimized the diameter of spheroids and the initiation timing for drug screening and emphasized the importance of AI-based morphological analysis in high-throughput screening.

Two-Cell Spheroid Angiogenesis Assay System Using Both Endothelial Colony Forming Cells and Mesenchymal Stem Cells

( Sajita Shah ),( Kyu-tae Kang ) 한국응용약물학회 2018 Biomolecules & Therapeutics(구 응용약물학회지) Vol.26 No.5

Most angiogenesis assays are performed using endothelial cells. However, blood vessels are composed of two cell types: endothelial cells and pericytes. Thus, co-culture of two vascular cells should be employed to evaluate angiogenic properties. Here, we developed an in vitro 3-dimensional angiogenesis assay system using spheroids formed by two human vascular precursors: endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs). ECFCs, MSCs, or ECFCs+MSCs were cultured to form spheroids. Sprout formation from each spheroid was observed for 24 h by real-time cell recorder. Sprout number and length were higher in ECFC+MSC spheroids than ECFC-only spheroids. No sprouts were observed in MSC-only spheroids. Sprout formation by ECFC spheroids was increased by treatment with vascular endothelial growth factor (VEGF) or combination of VEGF and fibroblast growth factor-2 (FGF-2). Interestingly, there was no further increase in sprout formation by ECFC+MSC spheroids in response to VEGF or VEGF+FGF-2, suggesting that MSCs stimulate sprout formation by ECFCs. Immuno-fluorescent labeling technique revealed that MSCs surrounded ECFC-mediated sprout structures. We tested vatalanib, VEGF inhibitor, using ECFC and ECFC+MSC spheroids. Vatalanib significantly inhibited sprout formation in both spheroids. Of note, the IC₅₀ of vatalanib in ECFC+MSC spheroids at 24 h was 4.0 ± 0.40 µM, which are more correlated with the data of previous animal studies when compared with ECFC spheroids (0.2 ± 0.03 µM). These results suggest that ECFC+MSC spheroids generate physiologically relevant sprout structures composed of two types of vascular cells, and will be an effective pre-clinical in vitro assay model to evaluate pro- or anti-angiogenic property.

Lethal Effects of Radiation and Platinum Analogues on Multicellular Spheroids of HeLa Cells

홍성언(Seong Eon Hong) 대한방사선종양학회 1989 Radiation Oncology Journal Vol.7 No.2

HeLa 세포의 Spheroid를 배양시켜 cis-platinum과 carboplatin으로 처리한 후 그 반응을 세포의 생존분획으로 분석하였다. 체외실험 model인 spheroid를 사용하여 platinum 유사체의 약효와 방사선 감수성을 평가하고 약제에 대한 단층 세포와 spheroid의 감수성 차이를 세포-생존곡선에서 규명하기 위하여 본 실험을 시행하였다. Cis-platinum 농도-곡선에서 spheroid의 Cq=3.4μM이고 Co=1.2μM이었다. 이것은 단충세포에 비하여 Co는 큰 변화가 없으나 Cq가 증가되어 cis-platinum이 저산소층 세포보다 활동적으로 분화하는 표면세포에 주로 작용하였으며, 반대로 carboplatin의 효과는 spheroid에 대한 (Co=15.0μM) 감수성이 단층세포 (Co=32.5μM)에 비하여 크게 증가되어, spheroid의 심층세포에 주로 작용하였다. 방사선과 carboplation의 병용효과를 세표 생존분획이 0.01 수준에서 isobologram으로 분석한 결과 상호작용으로 supra-additive 효과를 나타내었다. 따라서, carboplatin은 cis-platinum에 비하여 신장과 위장에 대한 독성작용이 적고, 방사선과 병용함으로써 더욱 효과적인 종양 치료에 중요한 역할을 할 것으로 기대한다. Multicellular tumor spheroids of HeLa cells have been grown in a stare culture system. Samples of spheroids were exposed for 2 h to graded concentration of cis-platinum and its analogue, carboplatin. and then response assayed by survival of clonogenic cells. The purpose of present experiment is to clarify the effectiveness of these platinum compounds and to evaluate intrinsic radiosensitivity of cells using spheroids of HeLa cells as an experimental in vitro model. Variations of the drug sensitivity of monolayers as well as spheroids were also evaluated in cell-survival curves. In cis-platinum concentration-survival curve, there was a large shoulder extending as far as Cq=3.4 μM, after which there was exponential decrease in survival curve having a Co Value of 1.2 μM in spheroids. While the Co for the spheroids was essentially no significant change, but Cq value was larger than that of monolayers. This suggest that the effect of cis-platinum is greater in the monolayer with actively proliferaing cells than hypoxic one. In the corboplatin concentration-survival curves, the Co value of spheroids was 15.0 μM and the ratio with the Co from monolayer cell (32.5 μM) was 0.46, thus indicating that the spheroids had a greater sensitivity to carboplatin than monolayers. Therefore, 'the effect of carboplatin is mainly on the deeper layers of spheroids acting as hypoxic cell sensitizer. The enhanced effect was obtained for monolayer cells using combined X-ray and carboplatin treatment 2 hours before irradiation. The result shown in isobologram analysis for the level of surviving fraction at 0.01 indicated that the effect of two agents was truely supra-additive. From this experimental data, carboplatin has excited much recent interest as one of the most promising, since it is almost without nephrotoxicity and causes less gastrointestinal toxicity than cis-platinurn. Interaction between carboplatin and radiation might play an important role for more effective local tumor control.

Size-controlled human adipose-derived stem cell spheroids hybridized with single-segmented nanofibers and their effect on viability and stem cell differentiation

Jin Kyu Lee,Sangmin Lee,Sungmin Kim,신흥수 한국생체재료학회 2021 생체재료학회지 Vol.25 No.2

Background: Fabrication of three-dimensional stem cell spheroids have been studied to improve stem cell function, but the hypoxic core and limited penetration of nutrients and signaling cues to the interior of the spheroid were challenges. The incorporation of polymers such as silica and gelatin in spheroids resulted in relatively relaxed assembly of composite spheroids, and enhancing transport of nutrient and biological gas. However, because of the low surface area between cells and since the polymers were heterogeneously distributed throughout the spheroid, these polymers cannot increase the cell to extracellular matrix interactions needed to support differentiation. Methods: We developed the stem cell spheroids that incorporate poly(ι-lactic acid) single-segmented fibers synthesized by electrospinning and physical and chemical fragmentation. The proper mixing ratio was 2000 cells/ μg fibers (average length of the fibers was 50 μm - 100 μm). The SFs were coated with polydopamine to increase cell binding affinity and to synthesize various-sized spheroids. The function of spheroids was investigated by in vitro analysis depending on their sizes. For statistical analysis, Graphpad Prism 5 software (San Diego, CA, USA) was used to perform one-way analysis of variance ANOVA with Tukey’s honest significant difference test and a Student’s t-test (for two variables) (P < 0.05). Results: Spheroids of different sizes were created by modulating the amount of cells and fibers (0.063mm2–0.322 mm2). The fibers in the spheroid were homogenously distributed and increased cell viability, while cell-only spheroids showed a loss of DNA contents, internal degradation, and many apoptotic signals. Furthermore, we investigated stemness and various functions of various-sized fiber-incorporated spheroids. In conclusion, the spheroid with the largest size showed the greatest release of angiogenic factors (released VEGF: 0.111 ± 0.004 pg/ng DNA), while the smallest size showed greater effects of osteogenic differentiation (mineralized calcium: 18.099 ± 0.271 ng/ng DNA). Conclusion: The spheroids incorporating polydopamine coated single-segmented fibers showed enhanced viability regardless of sizes and increased their functionality by regulating the size of spheroids which may be used for various tissue reconstruction and therapeutic applications.

Cellular context-dependent interaction between cancer and stellate cells in hetero-type multicellular spheroids of pancreatic tumor

Nam, Sanghun,Khawar, Iftikhar Ali,Park, Jong Kook,Chang, Suhwan,Kuh, Hyo-Jeong Elsevier 2019 Biochemical and biophysical research communication Vol.515 No.1

<P><B>Abstract</B></P> <P>Heterotypic cell-cell interaction between cancer cells and pancreatic stellate cells (PSCs) within tumor microenvironment is considered as a key mechanism for epithelial-mesenchymal transition (EMT) that triggers disease progression and chemoresistance in pancreatic ductal adenocarcinoma (PDAC). Hence, PSCs should be incorporated into <I>in vitro</I> co-culture models to maximize clinical relevance of data obtained using these models. In this study, we developed hetero-type spheroids of pancreatic cancer cells (ductal carcinoma cells PANC-1 and primacy sarcomatoid adenocarcinoma 36473 cells) and PSCs. Effect of PSC co-culture on the formation and growth of multicellular spheroids was cell-line dependent in that growth stimulation effect appeared in PANC-1/PSC spheroids, but not in 36473/PSC spheroids. Spatial distribution of PSCs within spheroids was also cell-line dependent. It was either confined to the center region (PANC-1) or evenly distributed (36473). Changes in expression levels of E-cadherin and vimentin revealed EMT induction in PANC-1/PSC hetero-type spheroids, but not in 36473/PSC spheroids. Gemcitabine sensitivity was increased partially by PSC co-culture. However, PSCs showed relative resistance to gemcitabine compared to PANC-1 cells in PANC-1/PSC spheroids. Overall, our hetero-type spheroid model can be used to study cancer-stroma interaction and their mechanism and evaluate anticancer drug activity. We demonstrated that stromal effect by PSC co-culture might be cellular context dependent with regard to growth stimulation and EMT induction. Hence, anti-stromal therapy should take these differences into consideration.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Hetero-type spheroids were established using pancreatic cancer cells (PANC-1 or 36473) and pancreatic stellate cells (PSCs). </LI> <LI> Growth stimulation effect and spatial distribution of PSCs in hetero-type spheroids were cell-line dependent. </LI> <LI> Changes in expression levels of EMT markers were observed in PANC-1/PSC hetero-type spheroids, but not in 36473/PSC spheroids. </LI> <LI> Gemcitabine sensitivity was increased partially by PSC co-culture in PANC-1 spheroids. </LI> </UL> </P>

Cryopreservable three-dimensional spheroid culture for ready-to-use systems

Thuy Trang Truong,이유빈,박경환,심혜은,송진정,김형선,황정호,강선웅,허강무 한국화학공학회 2023 Korean Journal of Chemical Engineering Vol.40 No.2

Three-dimensional (3D) spheroid culture has applications in many fields as spheroids closely recapitulate physiological conditions. However, spheroid culture and maintenance are time-consuming and unsuitable for urgent situations; therefore, appropriate cryopreservation methods for spheroids are required for their use in an on-demand and ready-to-use manner. We hypothesized that the feasibility of a ready-to-use system relies on diffusion of the preservation solution within spheroids; we thus evaluated the effects of spheroid-forming parameters, such as cell number and culture period, on spheroid viability and functionality. Long-term spheroid culture for seven days interfered with penetration of the cryopreservation solution as it caused cell condensation and extracellular matrix (ECM) secretion, as well as low viability and migratory activity upon replating after storage. However, ready-to-use spheroids, which were cultured for one day and then cryopreserved, showed viability and migration similar to those of non-cryopreserved spheroids, confirming that a short incubation period was suitable for this system. The chondrocyte-based ready-to-use spheroid system designed in this study can be easily applied to regenerative medicine applications that require a large number of cells in the future and can provide information for applying the ready-to-use spheroid system to various cell types.

한시적 세포포집 구조물을 이용한 다세포 스페로이드 형성 및 추출칩

진혜진(Hye-Jin Jin),김태윤(Taeyoon Kim),조영호(Young-Ho Cho),구진모(Jin-Mo Gu),김진국(Jhingook Kim),오용수(Yong-Soo Oh) 대한기계학회 2011 大韓機械學會論文集A Vol.35 No.2

본 논문에서는 한시적 세포포집 구조물을 이용한 다세포 스페로이드의 형성 및 추출칩을 제안하였다. 종래의 웰 플레이트와 플라스크는 작은 스페로이드 형성이 어렵고, 기존 마이크로칩은 고정된 세포포집 구조물을 이용하여 스페로이드 추출이 어려운 단점이 있다. 반면, 제안된 칩은 한시적 세포포집 구조물을 이용하여 스페로이드의 형성과 추출이 모두 용이한 장점이 있다. 50㎪ 의 박막압력으로 형성된 세포포집 구조물에 145~155㎩ 의 세포입력압력으로 유입되는 세포를 포집한 후, 24 시간 배양하여 스페로이드를 형성하였다. 또한, 박막압력 제거 후 5㎪ 의 세포입력압력으로 추출된 스페로이드의 지름과 활성도는 각각 197±11.7Bm, 80.3±7.7%로 측정되었다. 제안된 칩은 스페로이드의 균일한 형성과 안정적 추출이 용이하여 스페로이드의 후처리에 적용될 수 있다. We propose a spheroid chip that uses removable cell trapping barriers and that is capable of forming and extracting multicellular spheroids. By using a conventional well plate and flask, it is difficult to form small-sized spheroids, which resemble avascular 3D cell-cell interaction. It was difficult to extract spheroids using conventional microchips and fixed cell trapping barriers. The proposed chip, however, facilitates both formation and extraction of spheroids by using removable cell trapping barriers formed by membrane deflection. The cell trapping barriers, formed at the membrane pressure of 50 ㎪, hold the cells in the trapping region at a cell inlet pressure of 145?155 ㎩. After incubation for 24 h, the trapped cells form uniform spheroids. We successfully extract the spheroids at a cell inlet pressure of 5 ㎪ after removing the membrane pressure. The extracted spheroids have a diameter of 197.2 ± 11.7 Bm with a viability of 80.3 ± 7.7%. Using the proposed chip, uniform spheroids can be formed and these spheroids can be safely extracted for carrying out the post-processing of spheroids.