3D In Vitro Platform for Cell and Explant Culture in Liquid-like Solids

Abstract

Existing 3D cell models and technologies have offered tools to elevate cell culture to a more physiologically relevant dimension. One mechanism to maintain cells cultured in 3D is by means of perfusion. However, existing perfusion technologies for cell culture require complex electronic components, intricate tubing networks, or specific laboratory protocols for each application. We have developed a cell culture platform that simply employs a pump-free suction device to enable controlled perfusion of cell culture media through a bed of granular microgels and removal of cell-secreted metabolic waste. We demonstrated the versatile application of the platform by culturing single cells and keeping tissue microexplants viable for an extended period. The human cardiomyocyte AC16 cell line cultured in our platform revealed rapid cellular spheroid formation after 48 h and ~90% viability by day 7. Notably, we were able to culture gut microexplants for more than 2 weeks as demonstrated by immunofluorescent viability assay and prolonged contractility.

Keywords: 3D cell culture; in vitro; liquid-like solids; microexplants; microgels; perfusion.

Figure 1

24-Well Darcy Plate (A) A 24-well plate consists of 4 injection-molded polystyrene components: a lid, media reservoir, droplet-guiding cover, and effluent collection chambers. The lid protects the media reservoirs from sources of contamination and allows the exchange of gases. The media reservoir is divided into quadrants that each can contain up to 6 mL of culture media. (B) Detailed view of an individual well where samples can be suspended in LLS. A PCTE membrane is heat-sealed against the bottom surface during the plate assembly process, securing the suspension in place. A pressure device is used to create a light-vacuum pressure within the effluent collection compartments that drives the perfusion of media through the LLS and PCTE membrane.

Figure 2

Single-cell human cardiomyocytes cultured in 3D in LLS: (A) Phase contrast microscopy images of AC16 cells cultured in a 24-well Darcy plate at 0, 24, and 48 h showing cellular aggregation and spheroid formation. (B) Viability of AC16 spheroids on day 4 was assessed by fluorescent microscopy using Calcein AM (live) and BOBO-3 Iodide (dead). (C) Glucose consumption and (D) lactate secretion were measured from two technical replicates of daily effluent media collection, revealing metabolic activities of the AC16 cells. Statistical analysis was performed using two-way ANOVA with comparisons to the high flow condition to assess significance at each time point. (E,F) The percentage of cell viability was assessed by flow cytometry. (E) Characteristic flow profiles for all four conditions for one representative replicate on day 7 (typical for all samples, n = 3 replicates for each condition). The insets on the left show an example of a gating strategy used for assessing viability including the use of a heat-killed control. (F) Proportion of viable cells at day 0, day 3, and day 7 from different flow rates, 25.4 ± 4.1 (low), 35.3 ± 4.1 (medium), and 43.1 ± 4.8 µL/hr/well (high) for all samples, confirms significant viability of the cell populations in the presence of perfusion flow. Triplicate wells were used to measure viability in each condition, and two-way ANOVA with Dunnett’s post-test was used for statistical analysis.

Figure 3

Ex vivo culture of microexplants: (A) Diagram outlining major steps of mouse colorectal microtissue fabrication and culture in perfusion platform. (B) A series of time-lapse microscopy images showing the periodic contraction of a representative mouse colorectal microexplant from collected samples (n = 20) that had been cultured under perfusion for 9 h. Contraction frequency was estimated to be 3 contractions per minute (cpm) and was recorded at 1 fps for 10 min. (C) Assessment of microexplant contractility (4 ≤ n ≤ 9 per condition, * = p < 0.05) under 4 culture conditions: 3D hyperoxic (90% PO₂) and normoxic (20% PO₂), and 2D hyperoxic (90% PO₂) and normoxic (20% PO₂). Measurements were compared to the 2D 20% PO₂ to determine significance. (D) Micrographs of the viability of microexplants by fluorescent microscopy using Calcein AM (live) and BOBO-3 Iodide (dead). (E) Quantification of D (n = 3 per condition, * = p < 0.05, ** = p < 0.01, and *** = p < 0.001). (F) Representative microexplants from the 3D hyperoxic condition at days 8, 14, and 17, stained with Hoechst 33342 (nuclei), Alexa Fluor™ 568 Phalloidin (F-actin), and E-cadherin (epithelial cells), demonstrate the well-preserved heterogenous microexplant structure. Data in Figure 3 were collected from three separate mice and are representative of 5 experiments using 7 mice in total.