Gelatin maleimide microgels for hematopoietic progenitor cell encapsulation

Abstract

Hematopoietic stem cells (HSCs) are the apical cells of the hematopoietic system, giving rise to cells of the blood and lymph lineages. HSCs reside primarily within bone marrow niches that contain matrix and cell-derived signals that help inform stem cell fate. Aspects of the bone marrow microenvironment have been captured in vitro by encapsulating cells within hydrogel matrices that mimic native mechanical and biochemical properties. Hydrogel microparticles, or microgels, are increasingly being used to assemble granular biomaterials for cell culture and noninvasive delivery applications. Here, we report the optimization of a gelatin maleimide hydrogel system to create monodisperse gelatin microgels via a flow-focusing microfluidic process. We report characteristic hydrogel stiffness, stability, and swelling characteristics as well as encapsulation of murine hematopoietic stem and progenitor cells, and mesenchymal stem cells within microgels. Microgels support cell viability, confirming compatibility of the microfluidic encapsulation process with these sensitive bone marrow cell populations. Overall, this work presents a microgel-based gelatin maleimide hydrogel as a foundation for future development of a multicellular artificial bone marrow culture system.

Keywords: bone marrow; gelatin; hematopoietic stem cell; microgel.

Figure 1.

A Reaction scheme for conjugation of maleimide group to gelatin. NHS-Ester of N-hydroxysuccinimide 3-maleimidopropionate (1) reacts with primary amines of gelatin (2). B Overview of GelMAL production process, highlighting tuned reaction and dialysis steps. Created with Biorender.com. C Schematic diagram of a microfluidic device used to form cell-laden GelMAL microgels. An oil and an aqueous cell-containing GelMAL solution flow simultaneously through the flow-focusing nozzle into a DTT emulsion. Emulsified DTT diffuses into and crosslinks microgels. The microgels flow out of the device and are collected in PBS or cell media. D Still frame taken from video of microgel production. Scale bar 500 μm. E Image of microgels after washing. Scale bar = 200 μm.

Figure 2.

Coefficient of variation in microgel diameter vs. conjugation reaction pH. Each point corresponds to a single batch of GelMAL that was tested in microgel fabrication. All synthesis variations were dialyzed in distilled water. Inset: Representative photos and images of GelMAL precursor and washed microgels when using GelMAL synthesized below and above pH 5.9. Scale bar = 500 μm.

Figure 3.

Coefficient of variation in microgel diameter vs. dialysis pH, with representative images of microgel fabrication for each condition. All GelMAL batches were synthesized at pH 4.5. Scale bars = 500 μm. Bar plot reports mean ± standard error of the mean. Water dialysis: n = 3, pH 3.25 dialysis: n = 4. * denotes p < 0.05 (two-sample t-test).

Figure 4.

A Microgels were loaded into a microfluidic chip to measure microgel stability over a period of 4 days at 37 °C. Scale bar = 1 mm. B Fraction of microgels remaining after 4-day incubation. Bar plot reports mean ± standard error of the mean for n = 4 independent experiments. C % increase in microgel diameter by day 4, normalized to day 0 diameter from n = 4 independent experiments. Bar plot reports mean ± standard error of the mean for n = 4 independent experiments. D Compressive moduli of three batches of GelMAL, all synthesized by conjugating at pH 4.5 and dialyzing at pH 3.25. Bar plot reports mean ± standard error of the mean, and dots represent individual measured hydrogels. Minimum n = 4 for all batches.

Figure 5.

A Relative frequency of microgels containing x (0,1,2…) cells. Bars show the theoretical Poisson distribution for a specific cell density (Low, Med, High) in microgels of target diameter 145 μm. Lines show experimental values, with error bars showing standard deviation from the mean. B HSPCs and MSCs encapsulated inside of microgels without Optiprep and stained for viability. Blue = Cell (Hoechst 33342), Green = Live (Calcein), Red = Dead (BOBO-3). Scale bar = 50 μm. C Viability of unencapsulated cells (Control), and microgel-encapsulated HSPCs and MSCs. A minimum of 133 cells were measured for each of n = 3 technical replicates for HSPCs and a minimum of 152 cells were measured for each of n = 3 technical replicates for MSCs. Bars for microgel-encapsulated cells report the mean ± standard error of the mean. *** indicates p < 0.001 when compared against control (one-sample t-test).