Technical Advance: Novel ex vivo culture method for human monocytes uses shear flow to prevent total loss of transendothelial diapedesis function

Ex vivo culture conditions during the manufacturing process impact the therapeutic effect of cell-based products. Mimicking blood flow during ex vivo culture of monocytes has beneficial effects by preserving their migratory ability. However, the effects of shear flow on the inflammatory response have not been studied so far. Hence, the present study investigates the effects of shear flow on both blood-derived naïve and activated monocytes. The activation of monocytes was experimentally induced by granulocyte-macrophage colony-stimulating factor (GM-CSF), which acts as a pro-survival and growth factor on monocytes with a potential role in inflammation. Monocytes were cultured under dynamic (=shear flow) or static conditions while preventing monocytes' adherence by using cell-repellent surfaces to avoid adhesion-induced differentiation. After cultivation (40 h), cell size, viability, and cytokine secretion were evaluated, and the cells were further applied to functional tests on their migratory capacity, adherence, and metabolic activity. Our results demonstrate that the application of shear flow resulted in a decreased pro-inflammatory signaling concurrent with increased secretion of the anti-inflammatory cytokine IL-10 and increased migratory capacity. These features may improve the efficacy of monocyte-based therapeutic products as both the unwanted inflammatory signaling in blood circulation and the loss of migratory ability will be prevented.

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Ex Vivo Culture Models of Hidradenitis Suppurativa for defining molecular pathogenesis and treatment efficacy of novel drugs

10.1101/2021.10.11.464007 ◽

2021 ◽

Author(s):

Kayla Goliwas ◽

Mahendra P Kashyap ◽

Jasim Khan ◽

Rajesh Sinha ◽

Zhiping Weng ◽

...

Keyword(s):

Hidradenitis Suppurativa ◽

Ex Vivo ◽

Drug Efficacy ◽

Culture Method ◽

Gene Clusters ◽

Dynamic Expression ◽

Novel Drugs ◽

Culture Models ◽

Ex Vivo Culture ◽

Gene Status

Hidradenitis suppurativa (HS) is a complex inflammatory and debilitating skin disease for which no effective treatment is available. This is partly because of the unavailability of suitable human or animal models with which exact pathobiology of the disease can be defined. Here, we describe the development of air-liquid (A-L) interface, liquid-liquid/liquid-submersion (L-S) and bioreactor (Bio) ex vivo skin culture models. All three ex vivo platforms were effective for culturing skin samples up to day-14, with the tissue architecture and integrity remaining intact for at least 3 days for healthy skin while for 14 days for HS skin. Up to day-3, no significant differences were observed in % early apoptotic cells among all three platforms. However, an increase was observed in late apoptotic/necrotic cells in HS skin at day-3 in A-L and Bio culture of HS skin. These cultures efficiently support the growth of various cells populations, including keratinocytes and immune cells. Profiling of the inflammatory genes using HS skin from these ex vivo cultures showed dynamic expression changes at day-3 and day-14. All of these cultures are necessary to represent the inflammatory gene status of HS skin at day-0 suggesting that not all gene clusters are identically altered in each culture method. Similarly, cytokine/chemokine profiling of the supernatant from vehicle- and drug-treated ex vivo HS cultures again showed better prediction of drug efficacy against HS. Overall, development of these three systems collectively provide a powerful tool to uncover the pathobiology of HS progression and screen various drugs against HS.

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Novel Ex Vivo Culture Method for the Study of Dupuytren's Disease: Effects of TGFβ Type 1 Receptor Modulation by Antisense Oligonucleotides

Molecular Therapy — Nucleic Acids ◽

10.1038/mtna.2013.69 ◽

2014 ◽

Vol 3 ◽

pp. e142 ◽

Cited By ~ 13

Author(s):

Sofia Karkampouna ◽

Boudewijn PT Kruithof ◽

Peter Kloen ◽

Miryam C Obdeijn ◽

Annelies MA van der Laan ◽

...

Keyword(s):

Antisense Oligonucleotides ◽

Ex Vivo ◽

Culture Method ◽

Dupuytren’S Disease ◽

Dupuytren's Disease ◽

Receptor Modulation ◽

Ex Vivo Culture

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Ex Vivo Culture Models of Hidradenitis Suppurativa for Defining Molecular Pathogenesis and Treatment Efficacy of Novel Drugs

10.21203/rs.3.rs-1075860/v1 ◽

2021 ◽

Author(s):

Kayla Goliwas ◽

Mahendra P Kashyap ◽

Jasim Khan ◽

Rajesh Sinha ◽

Zhiping Weng ◽

...

Keyword(s):

Hidradenitis Suppurativa ◽

Ex Vivo ◽

Drug Efficacy ◽

Culture Method ◽

Gene Clusters ◽

Inflammatory Gene ◽

Dynamic Expression ◽

Culture Models ◽

Ex Vivo Culture ◽

Gene Status

Abstract Hidradenitis suppurativa (HS) is a complex inflammatory and debilitating skin disease for which no effective treatment is available currently. This is partly because of the lack of adequate human or animal models to define the pathobiology of the disease. Here, we describe the development of air-liquid (A-L) interface, liquid-submersion (L-S) and bioreactor (Bio) ex vivo skin culture models. All three ex vivo platforms were effective for culturing skin samples up to 14 days. Tissue architecture and integrity remained intact for at least 3 days for healthy skin and 14 days for HS skin. Up to day-3, no significant differences were observed in % early apoptotic cells among all three platforms. However, an increase was observed in late apoptotic/necrotic cells in HS skin at day-3 in A-L and Bio culture. These cultures efficiently support the growth of various cells populations, including keratinocytes and immune cells. Profiling inflammatory gene signatures in HS skin from these ex vivo cultures showed dynamic expression changes at day-3 and day-14. All three culture platforms are necessary to represent the inflammatory gene status of HS skin at day-0, suggesting that not all gene clusters are identically altered in each culture method. Similarly, cytokine/chemokine profiling of the supernatants from vehicle- and drug-treated ex vivo HS cultures again showed better prediction of drug efficacy against HS. Overall, development of these three culture systems collectively provides a powerful tool to uncover the pathobiology of HS progression and screen various drugs against HS.

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Personalized ex vivo culture method for analysis of antitumor treatment

Science-Business eXchange ◽

10.1038/scibx.2010.537 ◽

2010 ◽

Vol 3 (17) ◽

pp. 537-537

Keyword(s):

Ex Vivo ◽

Culture Method ◽

Ex Vivo Culture

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Phagocytosis of Liposome Particles by Rat Splenic Immature Monocytes Makes Them Transiently and Highly Immunosuppressive In Ex Vivo Culture Conditions

Journal of Pharmacology and Experimental Therapeutics ◽

10.1124/jpet.110.172510 ◽

2011 ◽

Vol 337 (1) ◽

pp. 42-49 ◽

Cited By ~ 8

Author(s):

Daisuke Takahashi ◽

Hiroshi Azuma ◽

Hiromi Sakai ◽

Keitaro Sou ◽

Daiko Wakita ◽

...

Keyword(s):

Ex Vivo ◽

Culture Conditions ◽

Ex Vivo Culture

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Stem cell integrins: Implications for ex-vivo culture and cellular therapies

Stem Cell Research ◽

10.1016/j.scr.2010.09.005 ◽

2011 ◽

Vol 6 (1) ◽

pp. 1-12 ◽

Cited By ~ 110

Author(s):

Andrew B.J. Prowse ◽

Fenny Chong ◽

Peter P. Gray ◽

Trent P. Munro

Keyword(s):

Stem Cell ◽

Ex Vivo ◽

Cellular Therapies ◽

Ex Vivo Culture

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Ex vivo culture of Fancc-/- stem/progenitor cells predisposes cells to undergo apoptosis, and surviving stem/progenitor cells display cytogenetic abnormalities and an increased risk of malignancy

Blood ◽

10.1182/blood-2004-06-2483 ◽

2005 ◽

Vol 105 (9) ◽

pp. 3465-3471 ◽

Cited By ~ 48

Author(s):

Xiaxin Li ◽

Michelle M. Le Beau ◽

Samantha Ciccone ◽

Feng-Chun Yang ◽

Brian Freie ◽

...

Keyword(s):

Progenitor Cells ◽

Genome Stability ◽

Bone Marrow Cells ◽

Ex Vivo ◽

Acquired Resistance ◽

Intrinsic Defects ◽

Cytogenetic Abnormalities ◽

Increased Risk ◽

Ex Vivo Culture ◽

The Impact

AbstractCurrent strategies for genetic therapy using Moloney retroviruses require ex vivo manipulation of hematopoietic cells to facilitate stable integration of the transgene. While many studies have evaluated the impact of ex vivo culture on normal murine and human stem/progenitor cells, the cellular consequences of ex vivo manipulation of stem cells with intrinsic defects in genome stability are incompletely understood. Here we show that ex vivo culture of Fancc-/- bone marrow cells results in a time-dependent increase in apoptosis of primitive Fancc-/- progenitor cells in conditions that promote the proliferation of wild-type stem/progenitor cells. Further, recipients reconstituted with the surviving Fancc-/- cells have a high incidence of cytogenetic abnormalities and myeloid malignancies that are associated with an acquired resistance to tumor necrosis factor α (TNF-α). Collectively, these data indicate that the intrinsic defects in the genomic stability of Fancc-/- stem/progenitor cells provide a selective pressure for cells that are resistant to apoptosis and have a propensity for the evolution to clonal hematopoiesis and malignancy. These studies could have implications for the design of genetic therapies for treatment of Fanconi anemia and potentially other genetic diseases with intrinsic defects in genome stability.

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