The microwell-mesh: A novel device and protocol for the high throughput manufacturing of cartilage microtissues

Futrega, Kathryn; Palmer, Jodie; Kinney, Mackenzie H.; Lott, William B.; Ungrin, Mark; Zandstra, Peter W.; Doran, Michael

doi:10.1016/j.biomaterials.2015.05.013

Cited by 74 publications

(157 citation statements)

References 28 publications

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“…All of the antibodies utilized in this placental MSC characterization process are listed in the Table of Materials/Equipment. Staining was performed as per the manufactures instructions, with analysis methods described here 19 .…”

Section: Characterization Of Placental Msc In Vitromentioning

confidence: 99%

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Pelekanos

Sardesai

Futrega

et al. 2016

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Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

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Section: Characterization Of Placental Msc In Vitromentioning

confidence: 99%

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Pelekanos

Sardesai

Futrega

et al. 2016

JoVE

Self Cite

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“…Controlling the specific size of microtissues enables optimisation of biological potency while generating a spheroidal cell product with a suitable diameter for injection via a minimally invasive needle or orthoscopic tool (Futrega et al 2016). Microtissue platforms enable efficient and rapid manufacture of thousands of multicellular microtissues of specifically controlled size Futrega et al 2015Futrega et al , 2017. Relative to single cell injections, microtissues may also reduce loss of cells from the site of delivery and deliver more mature and matrix-rich tissues.…”

Section: Electronic Supplementary Materialsmentioning

confidence: 99%

“…The number of potential therapeutic permutations and combinations is vast and as yet, there is no recognised gold standard. Studies that manipulate BMSC as multicellular spheroids or microtissues are increasingly common Baraniak and McDevitt 2012;Kabiri et al 2012;Futrega et al 2015Futrega et al , 2017, as 3D BMSC microtissue culture exhibit increased maintenance of stem-like qualities and improved differentiation capacity (Mendez-Ferrer et al 2010;Pinho et al 2013). Controlling the specific size of microtissues enables optimisation of biological potency while generating a spheroidal cell product with a suitable diameter for injection via a minimally invasive needle or orthoscopic tool (Futrega et al 2016).…”

Section: Electronic Supplementary Materialsmentioning

confidence: 99%

Bone marrow-derived stem/stromal cells (BMSC) 3D microtissues cultured in BMP-2 supplemented osteogenic induction medium are prone to adipogenesis

et al. 2018

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Bone marrow-derived mesenchymal stem/stromal cells (BMSC) may facilitate bone repair through secretion of factors that stimulate endogenous repair processes or through direct contribution to new bone through differentiation into osteoblast-like cells. BMSC microtissue culture and differentiation has been widely explored recently, with high-throughput platforms making large-scale manufacture of microtissues increasingly feasible. Bone-like BMSC microtissues could offer an elegant method to enhance bone repair, especially in small-volume non-union defects, where small diameter microtissues could be delivered orthoscopically. Using a high-throughput microwell platform, our data demonstrate that (1) BMSC in 3D microtissue culture result in tissue compaction, rather than growth, (2) not all mineralised bone-like matrix is incorporated in the bulk microtissue mass and (3) a significant amount of lipid vacuole formation is observed in BMSC microtissues exposed to BMP-2. These factors should be considered when optimising BMSC osteogenesis in microtissues or developing BMSC microtissue-based therapeutic delivery processes.

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“…Using flow cytometry, cells were characterized for their expression of CD44, CD90, CD73, CD105, CD146, CD45, CD34, HLA--DR, and tri--lineage differentiation capacity validated as described previously [352].…”

Section: Msc Isolation and Expansionmentioning

confidence: 99%

“…The PDMS plates were cured overnight in an 80°C oven. PDMS containing plates were sterilized by submerging in 70% ethanol for 1 hour, followed by repeated rinses of wells with PBS as described previously [352].…”

Section: Cd34mentioning

confidence: 99%

Device and application development for haematopoietic stem and progenitor cell (HSPC) and mesenchymal stromal cell (MSC) 3D spheroid cultures

Futrega¹

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KeywordsAIM 2: Utilization of the high throughput microwell platform to manufacture HSPC/MSC spheroids to improve the efficacy of direct BM transplantation.AIM 3: Development of an improved microwell platform that retains spheroids within discrete microwells throughout culture. AIM 4:Characterization of HSPC surface marker change in response to microwell material. DEVICE AND APPLICATION DEVELOPMENT FOR HSPC AND MSC 3D SPHEROID CULTURES iiiChapter 2 describes the development of a high throughput HSPC/MSC 3D spheroid co--culture embodied in AIM 1. I developed a high throughput polydimethylsiloxane (PDMS) microwell platform that enabled the manufacture of thousands of uniform 3D multicellular co--culture spheroids. Spheroids were assembled such that each contained 25--to--400 BM--derived MSC and 10 CB--derived CD34 + cells (a cell population enriched for HSPC). The outcomes from this Chapter guided the design of the subsequent three chapters. The specific outcomes and subsequent study results were as follows:Chapter 3: As described in Chapter 2, only modest improvements in expansion could be achieved by co--culturing HSPC in 3D MSC spheroids using a microwell platform. We reasoned that the 3D spheroids could, however, function as a biologically active anchor to improve direct BM transplantation outcomes. In this Chapter, we contrasted transplantation methods, including the use of MSC suspensions and 3D multicellular spheroids to modulate human CB--derived CD34 + cell retention within the BM cavity of NOD/SCID gamma (NSG) mice. Analysis revealed that the BM of injected femurs in some animals was saturated with human cells, while distal haematopoietic tissue engraftment was poor. These data show, for the first time, that retention of human haematopoietic cells within the BM enhances local engraftment at the expense of systemic engraftment.Chapter 4: As identified in Chapter 2, improvements in 3D spheroid co--cultures will require that the microwell platform is compatible with repeated medium exchange or dilution feeding. Multiple partial or complete medium exchanges can displace spheroids from discrete microwells, and this can result in either the loss of spheroids from culture, or spheroid amalgamation when displaced microtissues fall into common microwells. In this Chapter, I describe the first microwell platform that incorporates a mesh to retain microtissues within discrete microwells. We called this platform the microwell--mesh. We show that bonding a nylon mesh with an appropriate pore size over the microwell openings allows single cells to pass through the mesh into the microwells during the seeding process, but subsequently retains assembled microtissues within discrete microwells.Chapter 5: As identified in Chapter 2, the presence of PDMS, the material used to fabricate the microwell platform, altered CD38 surface expression on CB--derived CD34 + cells. Previous reports have shown that inhibition of retinoic acid signaling down--regulated CD38 iv DEVICE AND APPLICATION DEVELOPMENT FOR HSPC AND MSC 3D SPHE...

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The microwell-mesh: A novel device and protocol for the high throughput manufacturing of cartilage microtissues

Cited by 74 publications

References 28 publications

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Bone marrow-derived stem/stromal cells (BMSC) 3D microtissues cultured in BMP-2 supplemented osteogenic induction medium are prone to adipogenesis

Device and application development for haematopoietic stem and progenitor cell (HSPC) and mesenchymal stromal cell (MSC) 3D spheroid cultures

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