Thermoresponsive Inverted Colloidal Crystal Hydrogel Scaffolds for Lymphoid Tissue Engineering

Abstract

Inverted colloidal crystal (ICC) hydrogel scaffolds represent unique opportunities in modeling lymphoid tissues and expanding hematopoietic-lymphoid cells. Fully interconnected spherical pore arrays direct the formation of stromal networks and facilitate interactions between stroma and hematopoietic-lymphoid cells. However, due to the intricate architecture of these materials, release of expanded cells is restricted and requires mechanical disruption or chemical dissolution of the hydrogel scaffold. One potent biomaterials strategy to release pore-entrapped hematopoietic-lymphoid cells without breaking the scaffolds apart is to transiently increase the dimensions of these materials using stimuli-responsive polymers. Having this mindset, thermoresponsive ICC scaffolds that undergo rapid (<1 min) and substantial (>300%) diameter change over a physiological temperature range (4-37 °C) by using poly(N-isopropylacrylamide) (PNIPAM) with nanogel crosslinkers is developed. For a proof-of-concept study, the stromal niche by creating osteospheroids, aggregates of osteoblasts, and bone chips is first replicated, and subsequently Nalm-6 model hematopoietic-lymphoid cells are introduced. A sixfold increase in cell count is harvested when ICC hydrogel scaffolds are expanded without termination of the established 3D stromal cell culture. It is envisioned that thermoresponsive ICC hydrogel scaffolds will enable for scalable and sustainable ex vivo expansion of hematopoietic-lymphoid cells.

Keywords: PNIPAM; hydrogel scaffolds; inverted colloidal crystals; lymphoid tissue; stimuli-responsive materials.

Figure 1.

Fabrication of thermoresponsive ICC hydrogel scaffolds. a) Illustration of chemical structures during two-step polymerization to synthesize PNIPAM-NG. b) Schematic procedure of ICC PNIPAM-NG scaffold fabrication via application of PNIPAM-NG synthesis into colloidal crystal template-based ICC pocket scaffold fabrication. c) Optical microscopy images of thermoresponsive ICC PNIPAM-NG scaffolds (upper panels) and zoomed-in images of spherical pore arrays and interpore junctions (lower panels) at 4 °C and 37 °C. (d-e) Comparison of d) overall scaffold diameters (n = 6) and e) ICC pore diameters (n = 20) at 4 °C, room temperature (RT), and 37 °C, respectively. (*P < 0.05)

Figure 2.

Characterization of the mechanical and optical properties of PNIPAM hydrogels. a) Compressive Young’s modulus of ICC PNIPAM-NG at 4 °C, RT, and 37 °C (n = 3). b) Relative optical transparency of ICC PNIPAM-NG based on water (set as 1) at 4 °C, RT, and 37 °C (n = 3). c) Compositions of four different PNIPAM hydrogels with schematic illustrations. (d-e) Comparison of the characteristic Young’s modulus at RT d) between Bulk PNIPAM-NG and Bulk PNIPAM (n = 5), and e) between ICC PNIPAM-NG and ICC PNIPAM (n = 5). (*P < 0.05).

Figure 3.

Comparative characterization of temperature-dependent volume changes in PNIPAM hydrogels. a) Snapshots of Bulk PNIPAM, Bulk PNIPAM-NG, ICC PNIPAM, and ICC PNIPAM-NG at discrete time points in pure water during 3 cycles of steady temperature change between 4 °C and 50 °C at 1 °C per min. b) Quantitative representation of temperature cycles (purple) and normalized deswelling ratios of Bulk PNIPAM (black), Bulk PNIPAM-NG (green), ICC PNIPAM (blue), and ICC PNIPAM-NG (red) with respect to time. (D₀ = initial hydrogel diameter at 4 °C, D_t = hydrogel diameter at a given temperature). c) Characteristic hysteresis curves of Bulk PNIPAM, Bulk PNIPAM-NG, ICC PNIPAM, and ICC PNIPAM-NG as a function of temperature and normalized swelling ratio during 3 cycles of steady temperature change. Arrows identify two distinct kinetic regimes for the temperature-dependent normalized deswelling ratios. Dotted lines represent critical temperatures in which deswelling ratio changes significantly. d) Comparison of thermoresponsive kinetics of ICC PNIPAM-NG under sudden (left) and steady (right) temperature changes between 4 °C and 37 °C. e) Schematic explaining the differences in response rate between Bulk PNIPAM-NG and ICC PNIPAM-NG as a function of mass transport. Mass transport in ICC PNIPAM-NG is considerably faster than bulk PNIPAM-NG as ICC geometry facilitates convective transport whereas bulk hydrogels only support transport via diffusion.

Figure 4.

Creation of osteospheroids in thermoresponsive ICC hydrogel scaffolds. a) Schematic of isolation and expansion of osteoblastic cells from a DsRed transgenic reporter mouse (left) and fluorescent microscopy image of DsRed osteoblasts on tissue culture plastic surface (right). b) Gross image of bovine trabecular bone (left) and optical microscopy image of bone chips after mechanical grinding (right). c) Comparison of osteoblast seeding efficiency in ICC PNIPAM-NG scaffolds between 4 °C and 37 °C. (n = 6). d) Schematic procedure of creating osteospheroids consisting of osteoblasts and bone chips in ICC PNIPAM-NG. (e-f) Confocal microscopy images e) after osteoblast seeding (left) and zoomed-in images of ICC pores with and without bone chips (left) and f) after 1 week of culture with and without bone chips (right). g) Quantitative comparison of osteospheroid diameters at 1 and 3 weeks of culture in ICC PNIPAM-NG as a function of increasing bone chip concentration (n = 5). (N.S.: not significant, *P < 0.05)

Figure 5.

Expansion and retrieval of Nalm-6 model hematopoietic-lymphoid cells in ICC PNIPAM-NG scaffolds co-cultured with osteospheroids. a) Schematic procedure of experiment to seed, expand, and retrieve GFP Nalm-6 model hematopoietic-lymphoid cells in ICC PNIPAM-NG scaffolds. b) Confocal 3D microscope images of expanding GFP Nalm-6 cells in ICC pores with co-cultured osteospheroids and subsequent in situ densification during 1-week of culture. c) Fold increase of Nalm-6 cells retrieved after 1-week culture in ICC PNIPAM-NG hydrogel scaffolds by leveraging its thermoresponsive properties and rapid response rate. (*P < 0.05)