Microliters Of Medium Research Articles

Chondrogenesis of Adipose-Derived Stem Cells Using an Arrayed Spheroid Format.

The chondrogenic response of adipose-derived stem cells (ASCs) is often assessed using 3D micromass protocols that use upwards of hundreds of thousands of cells. Scaling these systems up for high-throughput testing is technically challenging and wasteful given the necessary cell numbers and reagent volumes. However, adopting microscale spheroid cultures for this purpose shows promise. Spheroid systems work with only thousands of cells and microliters of medium. Molded agarose microwells were fabricated using 2% w/v molten agarose and then equilibrated in medium prior to introducing cells. ASCs were seeded at 50, 500, 5k cells/microwell; 5k, 50k, cells/well plate; and 50k and 250k cells/15mL centrifuge tube to compare chondrogenic responses across spheroid and micromass sizes. Cells were cultured in control or chondrogenic induction media. ASCs coalesced into spheroids/pellets and were cultured at 37°C and 5% CO2 for 21days with media changes every other day. All culture conditions supported growth of ASCs and formation of viable cell spheroids/micromasses. More robust growth was observed in chondrogenic conditions. Sulfated glycosaminoglycans and collagen II, molecules characteristics of chondrogenesis, were prevalent in both 5000-cell spheroids and 250,000-cell micromasses. Deposition of collagen I, characteristic of fibrocartilage, was more prevalent in the large micromasses than small spheroids. Chondrogenic differentiation was consistently induced using high-throughput spheroid formats, particularly when seeding at cell densities of 5000 cells/spheroid. This opens possibilities for highly arrayed experiments investigating tissue repair and remodeling during or after exposure to drugs, toxins, or other chemicals. The online version contains supplementary material available at 10.1007/s12195-022-00746-8.

Tracking leukemic T-cell transcriptional dynamics in vivo with a blood-based reporter assay.

Transcriptional dynamics of cancer cells govern cell fate decisions and are therapeutically actionable drug targets. In this study, we engineered a circulating cancer cell line that secretes a luciferase reporter to capture constitutive and circadian clock-driven transcription dynamics over the course of a day. Engineered human leukemic T cells (Jurkat) were observed to rhythmically secrete luciferase in a continuous flow cell culture system. When transplanted invivo, engineered leukemic cells caused circadian plasma luciferase activity and had expected pathological signs of leukemic disease. This technique is rapid and noninvasive, requiring only a few microliters of media or blood, and can aid in investigating relationships between invivo cancer cell signaling and behavior, such as diet or sleep.

Effect of hCG on protein synthesis and progesterone production in the bovine luteal cells.

The study was carried out to investigate whether the luteal steroidogenesis in response to tropic stimulus is mediated through de novo synthesis of protein(s). Luteal cell suspension was prepared by collagenase-DNAse treatment of the bovine corpora lutea, weighing 4-6 g. Cells equivalent to 250 micrograms of protein were incubated in a total volume of 550 microliter of medium 199 with or without various test substances. Production of progesterone by the luteal cells was stimulated by hCG in a dose dependent manner. Incorporation of 3H-leucine into the cellular proteins was concomitantly enhanced. Emetine, cycloheximide and puromycin inhibited both basal and hCG stimulated protein synthesis as well as progesterone production in the cells. Thus, luteal steroidogenesis induced by hCG seems to be dependent upon de novo synthesis of a protein(s).

Cultured rat trachea as a model for the study of ciliary abundance: the effect of oxygen.

Cultured rat trachea as a model for the study of ciliary abundance: the effect of oxygen.