Dissociation of mono- and co-culture spheroids into single cells for subsequent flow cytometric analysis

Grässer, Ute; Bubel, Monika; Sossong, Daniela; Oberringer, Martin; Pohlemann, Tim; Metzger, Wolfgang

doi:10.1016/j.aanat.2017.10.002

Cited by 25 publications

(25 citation statements)

References 29 publications

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“…This could indicate that co‐cultured spheroids proliferate and mimic better the in vivo growth situation of heterocellular tissues with increasing culture time. This was in line with former studies reporting that mono‐cultured spheroids were lower in diameter versus co‐cultured spheroids (Grasser et al, 2018). This was clearly observed when adding HUVEC to the ASC spheroids and the co‐culture spheriods increased in diameter compared with the mono‐culture spheroids, with no difference in diameter between the two co‐culture conditions (VAT‐2S).…”

Section: Discussionsupporting

confidence: 92%

High‐throughput fabrication of vascularized adipose microtissues for 3D bioprinting

Benmeridja

Moor

Maere

et al. 2020

J Tissue Eng Regen Med

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For patients with soft tissue defects, repair with autologous in vitro engineered adipose tissue could be a promising alternative to current surgical therapies. A volumepersistent engineered adipose tissue construct under in vivo conditions can only be achieved by early vascularization after transplantation. The combination of 3D bioprinting technology with self-assembling microvascularized units as building blocks can potentially answer the need for a microvascular network. In the present study, co-culture spheroids combining adipose-derived stem cells (ASC) and human umbilical vein endothelial cells (HUVEC) were created with an ideal geometry for bioprinting. When applying the favourable seeding technique and condition, compact viable spheroids were obtained, demonstrating high adipogenic differentiation and capillary-like network formation after 7 and 14 days of culture, as shown by live/dead analysis, immunohistochemistry and RT-qPCR. Moreover, we were able to successfully 3D bioprint the encapsulated spheroids, resulting in compact viable spheroids presenting capillary-like structures, lipid droplets and spheroid outgrowth after 14 days of culture. This is the first study that generates viable high-throughput (pre-)vascularized adipose microtissues as building blocks for bioprinting applications using a novel ASC/HUVEC co-culture spheroid model, which enables both adipogenic differentiation while simultaneously supporting the formation of prevascular-like structures within engineered tissues in vitro. K E Y W O R D S

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Section: Discussionsupporting

confidence: 92%

High‐throughput fabrication of vascularized adipose microtissues for 3D bioprinting

Benmeridja

Moor

Maere

et al. 2020

J Tissue Eng Regen Med

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“…The LF spheroids were first dissociated following the method reported by Grasser et al [26] Several spheroids were collected from a ULA 96-well plate in a centrifuge tube (15 mL). After precipitation of the spheroids, the supernatants were carefully removed and washed twice with 1× PBS.…”

Section: Flow Cytometrymentioning

confidence: 99%

Development of an In Vitro 3D Model for Investigating Ligamentum Flavum Hypertrophy

et al. 2020

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Background: Ligamentum flavum hypertrophy (LFH) is among the most crucial factors in degenerative lumbar spinal stenosis, which can cause back pain, lower extremity pain, cauda equina syndrome and neurogenic claudication. The exact pathogenesis of LFH remains elusive despite extensive research. Most in vitro studies investigating LFH have been carried out using conventional two-dimensional (2D) cell cultures, which do not resemble in vivo conditions, as they lack crucial pathophysiological factors found in three-dimensional (3D) LFH tissue, such as enhanced cell proliferation and cell cluster formation. In this study, we generated ligamentum flavum (LF) clusters using spheroid cultures derived from primary LFH tissue. Results: The cultured LF spheroids exhibited good viability and growth on an ultra-low attachment 96-well plate (ULA 96-plate) platform according to live/dead staining. Our results showed that the 100-cell culture continued to grow in size, while the 1000-cell culture maintained its size, and the 5000-cell culture exhibited a decreasing trend in size as the culture time increased; long-term culture was validated for at least 28 days. The LF spheroids also maintained the extracellular matrix (ECM) phenotype, i.e., fibronectin, elastin, and collagen I and III. The 2D culture and 3D culture were further compared by cell cycle and Western blot analyses. Finally, we utilized hematoxylin and eosin (H&E) staining to demonstrate that the 3D spheroids resembled part of the cell arrangement in LF hypertrophic tissue. Conclusions: The developed LF spheroid model has great potential, as it provides a stable culture platform in a 3D model that can further improve our understanding of the pathogenesis of LFH and has applications in future studies.

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“…Single cells within spheroids were analyzed by flow cytometry. 23 Briefly, spheroids were washed with PBS and dissociated in a solution of 0.1% (w/v) type I collagenase (Calbiochem, San Diego, California) and 0.3 U dispase (Thermo Fisher) at 37 C with gentle external agitation. After 1 hour, an equal volume of media was added to quench enzymatic activity, and cells were washed with PBS.…”

Section: Flow Cytometrymentioning

confidence: 99%

Tunneling nanotubes mediate the expression of senescence markers in mesenchymal stem/stromal cell spheroids

et al. 2019

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The therapeutic potential of mesenchymal stem/stromal cells (MSCs) is limited by acquired senescence following prolonged culture expansion and high-passage numbers.However, the degree of cell senescence is dynamic, and cell-cell communication is critical to promote cell survival. MSC spheroids exhibit improved viability compared with monodispersed cells, and actin-rich tunneling nanotubes (TNTs) may mediate cell survival and other functions through the exchange of cytoplasmic components. Building upon our previous demonstration of TNTs bridging MSCs within these cell aggregates, we hypothesized that TNTs would influence the expression of senescence markers in MSC spheroids. We confirmed the existence of functional TNTs in MSC spheroids formed from low-passage, high-passage, and mixtures of low-and high-passage cells using scanning electron microscopy, confocal microscopy, and flow cytometry. The contribution of TNTs toward the expression of senescence markers was investigated by blocking TNT formation with cytochalasin D (CytoD), an inhibitor of actin polymerization. CytoD-treated spheroids exhibited decreases in cytosol transfer. Compared with spheroids formed solely of high-passage MSCs, the addition of low-passage MSCs reduced p16 expression, a known genetic marker of senescence. We observed a significant increase in p16 expression in high-passage cells when TNT formation was inhibited, establishing the importance of TNTs in MSC spheroids. These data confirm the restorative role of TNTs within MSC spheroids formed with low-and high-passage cells and represent an exciting approach to use higher-passage cells in cell-based therapies. K E Y W O R D Smesenchymal stem/stromal cells, p16, senescence, spheroids, tunneling nanotubes

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Dissociation of mono- and co-culture spheroids into single cells for subsequent flow cytometric analysis

Cited by 25 publications

References 29 publications

High‐throughput fabrication of vascularized adipose microtissues for 3D bioprinting

High‐throughput fabrication of vascularized adipose microtissues for 3D bioprinting

Development of an In Vitro 3D Model for Investigating Ligamentum Flavum Hypertrophy

Tunneling nanotubes mediate the expression of senescence markers in mesenchymal stem/stromal cell spheroids

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