(M1130-04-24) Collagen Droplet-Based Micro-Bioreactors for High-Throughput Drug Screening

Purpose: The tumor microenvironment (TME) is known for its complexity, which is typically comprised by heterogeneous cell populations in three-dimensional arrangement for playing a dynamic role in tumorigenesis. In order to harness the intrinsic characteristics of such tumor microenvironment and effectively screen therapeutic drug function, in vitro tumor models are commonly developed. However, due to the lack of cellular communication and cell-matrix interactions, 2D cell culture has been proved for being unable to reflect the in vivo tumor status. Recently, 3D spheroid culture has been intensively developed for capturing the physico-biochemical characteristics of TME. Although, the throughput for large-scale drug screening using 3D spheroids is still limited. In this research, we introduce a high throughput, customizable, large-scale 3D spheroid generation platform using novel microfluidic collagen droplets, which produces ~2x10⁴ 3D spheroids as individual micro-bioreactors in one hour, as well as for bio-inks utilized in extrusion-based 3D bioprinting. Tunable cell encapsulated collagen droplets act as micro-bioreactors to facilitate optimal growth of tumor spheroids to be infused into a biopolymer-based scaffold to further facilitate cell-adhesion and growth in natural occurring architectures mimicking TME.

Methods: An array of microfluidic droplet generators was designed using AutoCAD software and fabricated using 3D printing and molding technology (Fig 1.A). The device consists of two phases including oil and aqueous collagen. The devices were cast in PDMS and bonded to a glass slide using oxygen plasma. Bovine type I collagen was diluted to a concentration of 0.5 mg/ml and mixed with GFP expressing H322 bronchoalveolar carcinoma cells (Fig 1 B). Controlling the microfluidic droplet platform at 4 C could maintain fluidic properties of collagen for high-throughput, continuous collagen-in-oil droplet generation with encapsulated cells (Fig. 1 B). While harvesting collagen-in-oil droplets and bringing the temperature to 37 C, collagen-in-oil droplets could turn into solid due to the cross-link of collagen material. Different sizes of droplets were generated to optimize the encapsulation rate of cells from single-cell to multiple cells aggregation. Encapsulated cells in collagen droplets can be continuously cultured to grow into spheroids in size of ~500 microns within 5-day incubation (Fig. 1B). Afterward, cell-encapsulated collagen droplets can be introduced into a hydrogel mixture of 7% gelatin type A and 3.5% of sodium alginate for 3D printing to mimic in vivo tissue or organ structure using EnvisionTEC 3D Bioplotter Fig. 1. C- E)

Results: At 4 ⁰C, liquid collagen can form droplets that encapsulate cells, and these droplets form crosslinked solid sphere structure at 37 ⁰C under culture conditions. We used an oil phase and aqueous phase pressure ratio of 4:1 psi, which yielded droplet sizes of approximately 400 μm (Fig. 1B)We then experimented with different collagen concentrations to identify the most stable and durable matrix for cell seeding and found that 0.5 mg/ml provided optimal durability and permeability. We subsequently evaluated the correlation between cell concentration and growth rate within the droplet. Our findings demonstrated that successful spheroidal growth was achieved at a density of approximately 25 × 10⁶ cells/mL. Spheroids were then incubated for 4 days until confluency was reached (Fig.1 B). Lastly, we used the spheroids as a bio-ink supplemented with gelatin and alginate to print an alveoli-like tissue structure. The printing process was conducted at 28 ⁰C and involved constructing a 5-layer pattern for a lung-like tissue scaffold. We then monitored cell migration and growth within the scaffold.

Conclusion: In summary, we demonstrated using collagen as an essential biomaterial to generate robust spheroidal tumor TME in high throughput with microfluidic collagen droplet generator. The growth of these tumor spheroids can be influenced by several factors, such as cell density, flow velocity phase, and permeability of the droplets. 3D tumor encapsulated collagen droplets could be effective microbioreactors for large-scale drug candidate screening. The use of collagen matrix spheroids as bio-ink for extrusion-based 3D bioprinting has proven to be an effective method for fabricating in vivo-like 3D lung tumor models. This approach can provide high shape fidelity for lung tumor models, which is beneficial in testing various treatments and drugs for lung cancer.
Figure 1. Collagen droplet based bioreactor schematic.
A. Microfluidic device was modeled in AutoCAD and fabricated using 3D printing and soft-litography. This microfluidic platform was used to flow collagen suspension at 4°C and FC40 oil at 37°C to generate crosslinked collagen droplets B. H322 cells were mixed in with Collagen Type I with at concentrations of 0.5 mg/ml. Then cells were encapsulated within collagen droplets using microfluidic device generating 3D spheroids. Spheroidal growth was monitored and recorded. C After encapsulation and subsequent incubation, spheroids were mixed into gelatin and alginate hydrogel. Then using an extrusion-based 3D printer scaffolds were produced. D. Scaffolds containing H322 spheroids were printed and crosslinked using CaCl2. E. These were then incubated, and cell growth was monitored.