Dataset Information

Ultrahigh-throughput Generation and Characterization of Cellular Aggregates in Laser-ablated Microwells of Poly(dimethylsiloxane).

ABSTRACT: Aggregates of cells, also known as multicellular aggregates (MCAs), have been used as microscale tissues in the fields of cancer biology, regenerative medicine, and developmental biology for many decades. However, small MCAs (fewer than 100 cells per aggregate) have remained challenging to manufacture in large quantities at high uniformity. Forced aggregation into microwells offers a promising solution for forming consistent aggregates, but commercial sources of microwells are expensive, complicated to manufacture, or lack the surface packing densities that would significantly improve MCA production. To address these concerns, we custom-modified a commercial laser cutter to provide complete control over laser ablation and directly generate microwells in a poly(dimethylsiloxane) (PDMS) substrate. We achieved ultra rapid microwell production speeds (>50,000 microwells/hr) at high areal packing densities (1,800 microwells/cm2) and over large surface areas for cell culture (60 cm2). Variation of the PDMS substrate distance from the laser focal plane during ablation allowed for the generation of microwells with a variety of sizes, contours, and aspect ratios. Casting of high-fidelity microneedle masters in polyurethane allowed for non-ablative microwell reproduction through replica molding. MCAs of human bone marrow derived mesenchymal stem cells (hMSCs), murine 344SQ metastatic adenocarcinoma cells, and human C4-2 prostate cancer cells were generated in our system with high uniformity within 24 hours, and computer vision software aided in the ultra-high-throughput analysis of harvested aggregates. Moreover, MCAs maintained invasive capabilities in 3D migration assays. In particular, 344SQ MCAs demonstrated epithelial lumen formation on Matrigel, and underwent EMT and invasion in the presence of TGF-?. We expect this technique to find broad utility in the generation and cultivation of cancer cell aggregates, primary cell aggregates, and embryoid bodies.

SUBMITTER: Albritton JL

PROVIDER: S-EPMC4792302 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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Ultrahigh-throughput Generation and Characterization of Cellular Aggregates in Laser-ablated Microwells of Poly(dimethylsiloxane).

Albritton Jacob L JL Roybal Jonathon D JD Paulsen Samantha J SJ Calafat Nick N Flores-Zaher Jose A JA Farach-Carson Mary C MC Gibbons Don L DL Miller Jordan S JS

RSC advances 20160112 11

Aggregates of cells, also known as multicellular aggregates (MCAs), have been used as microscale tissues in the fields of cancer biology, regenerative medicine, and developmental biology for many decades. However, small MCAs (fewer than 100 cells per aggregate) have remained challenging to manufacture in large quantities at high uniformity. Forced aggregation into microwells offers a promising solution for forming consistent aggregates, but commercial sources of microwells are expensive, complic ...[more]

PMID: 26998251

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Project description:A method to efficiently immobilize and partition large quantities of microbeads in an array format in microfabricated poly(dimethylsiloxane) (PDMS) cassette for ultrahigh-throughput in situ releasable solution-phase cell-based screening of one-bead-one-compound (OBOC) combinatorial libraries is described. Commercially available Jeffamine triamine T-403 (∼440 Da) was derivatized such that two of its amino groups were protected by Fmoc and the remaining amino group capped with succinic anhydride to generate a carboxyl group. This resulting trifunctional hydrophilic polymer was then sequentially coupled two times to the outer layer of topologically segregated bilayer TentaGel (TG) beads with solid phase peptide synthesis chemistry resulting in beads with increased loading capacity, hydrophilicity, and porosity at the outer layer. We have found that such bead configuration can facilitate ultrahigh-throughput in situ releasable solution-phase screening of OBOC libraries. An encoded releasable OBOC small molecule library was constructed on Jeffamine derivatized TG beads with library compounds tethered to the outer layer via a disulfide linker and coding tags in the interior of the beads. Compound-beads could be efficiently loaded (5-10 min) into a 5 cm diameter Petri dish containing a 10,000-well PDMS microbead cassette, such that over 90% of the microwells were each filled with only one compound-bead. Jurkat T-lymphoid cancer cells suspended in Matrigel were then layered over the microbead cassette to immobilize the compound-beads. After 24 h of incubation at 37 °C, dithiothreitol was added to trigger the release of library compounds. Forty-eight hours later, MTT reporter assay was used to identify regions of reduced cell viability surrounding each positive bead. From a total of about 20,000 beads screened, 3 positive beads were detected and physically isolated for decoding. A strong consensus motif was identified for these three positive compounds. These compounds were resynthesized and found to be cytotoxic (IC(50) 50-150 μM) against two T-lymphoma cell lines and less so against the MDA-MB 231 breast cancer cell line. This novel ultrahigh-throughput OBOC releasable method can potentially be adapted to many existing 96- or 384-well solution-phase cell-based or biochemical assays.

Project description:Growth factors have been shown to be potent mediators of osteogenesis. However, their use in tissue-engineered scaffolds not only can be costly but also can induce undesired responses in surrounding tissues. Thus, the ability to specifically induce osteogenic differentiation in the absence of exogenous growth factors through manipulation of scaffold material properties would be desirable for bone regeneration. Previous research indicates that addition of inorganic or hydrophobic components to organic, hydrophilic scaffolds can enhance multipotent stem cell (MSC) osteogenesis. However, the combined impact of scaffold inorganic content and hydrophobicity on MSC behavior has not been systematically explored, particularly in three-dimensional (3D) culture systems. The aim of the present study was therefore to examine the effects of simultaneous increases in scaffold hydrophobicity and inorganic content on MSC osteogenic fate decisions in a 3D culture environment toward the development of intrinsically osteoinductive scaffolds. Mouse 10T½ MSCs were encapsulated in a series of novel scaffolds composed of varying levels of hydrophobic, inorganic poly(dimethylsiloxane) (PDMS) and hydrophilic, organic poly(ethylene glycol) (PEG). After 21 days of culture, increased levels of osteoblast markers, runx2 and osteocalcin, were observed in scaffolds with increased PDMS content. Bone extracellular matrix (ECM) molecules, collagen I and calcium phosphate, were also elevated in formulations with higher PDMS:PEG ratios. Importantly, this osteogenic response appeared to be specific in that markers for chondrocytic, smooth muscle cell, and adipocytic lineages were not similarly affected by variations in scaffold PDMS content. As anticipated, the increase in scaffold hydrophobicity accompanying increasing PDMS levels was associated with elevated scaffold serum protein adsorption. Thus, scaffold inorganic content combined with alterations in adsorbed serum proteins may underlie the observed cell behavior.

Dataset Information

Ultrahigh-throughput Generation and Characterization of Cellular Aggregates in Laser-ablated Microwells of Poly(dimethylsiloxane).

Publications

Ultrahigh-throughput Generation and Characterization of Cellular Aggregates in Laser-ablated Microwells of Poly(dimethylsiloxane).

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