Assessing the compatibility of primary human hepatocyte culture within porous silk sponges

Abstract

Donor organ shortages have prompted the development of alternative implantable human liver tissues for patients suffering from end-stage liver failure. Purified silk proteins provide desirable features for generating implantable tissues, including sustainable sourcing from insects/arachnids, biocompatibility, tunable mechanical properties and degradation rates, and low immunogenicity upon implantation. While different cell types were previously cultured for weeks within silk-based scaffolds, it remains unclear whether such scaffolds can be used to culture primary human hepatocytes (PHH) isolated from livers. Therefore, here we assessed the compatibility of PHH culture within porous silk scaffolds that enable diffusion of oxygen/nutrients through the pores. We found that incorporation of type I collagen during the fabrication and/or autoclaving of porous silk scaffolds, as opposed to simple adsorption of collagen onto pre-fabricated silk scaffolds, was necessary to enable robust PHH attachment/function. Scaffolds with small pores (73 ± 25 μm) promoted larger PHH spheroids and consequently higher PHH functions than large pores (235 ± 84 μm) for at least 1 month in culture. Further incorporation of supportive fibroblasts into scaffolds enhanced PHH functions up to 5-fold relative to scaffolds with PHHs alone and 2D co-cultures on plastic. Lastly, encapsulating PHHs within protein hydrogels while housed in the silk scaffold led to higher functions than protein hydrogel-only or silk-only controls. In conclusion, porous silk scaffolds containing extracellular matrix proteins can be used for the culture of PHHs ± supportive non-parenchymal cells, which can be further built on in the future to create optimized silk-based liver tissue surrogates for cell-based therapy.

Fig. 1. Culture of cells within porous silk sponges (scaffolds). Left to right: Insoluble silk scaffolds containing type I collagen (from rat tails) were fabricated and autoclaved per the protocols described in methods and then partially dehydrated by manually squeezing the hepatocyte culture medium out of the pores. The scaffolds were subsequently placed within the wells of a 48-well plate which was pre-coated with Pluronic F-127 to prevent cell attachment to the plastic. A cell suspension of PHHs ± 3T3-J2 fibroblasts was seeded onto the partially dehydrated silk scaffolds, which wicked up the cell suspension and allowed cells to attach over 3–4 hours at 37 °C to the collagen within the silk scaffolds. Once cells attached, additional cell culture medium was added to the wells and replaced every 4 days with fresh medium. Culture medium was collected for supernatant-based assays and the cell-laden scaffolds were also fixed at specific time-points for immunostaining of hepatic markers. PHHs stained with a human albumin antibody are shown to the right. Top row scale bar is 100 μm and bottom row scale bars are 5 mm.

Fig. 2. PHH distribution within composite silk-collagen I scaffolds of small or large pore sizes. Silk scaffolds containing collagen I were fabricated with (A) small pores (73 ± 25 μm) or (B) large pores (235 ± 84 μm) and then PHHs were seeded into the scaffolds per the protocols described in methods. After 3 weeks, the cell-laden scaffolds were fixed, sectioned, and stained for intracellular albumin. Shown in both panels is a single section and magnified images from three different regions within the scaffold to show the distribution of PHH attachment. The scale bars in magnified images are 250 μm.

Fig. 3. PHH functions within silk/collagen scaffolds of different pore sizes. Collagen I was incorporated into silk scaffolds by either autoclaving lyophilized silk with a 1 mg mL⁻¹ collagen in PBS solution (‘Silk + Autoclaved Collagen’) or by mixing the solubilized silk solution with 1 mg mL⁻¹ collagen in PBS followed by lyophilization and then further autoclaving in 1 mg mL⁻¹ collagen in PBS (‘collagen incorporated silk + autoclaved collagen’). PHHs were seeded into the scaffolds as described in methods. (A) PHH albumin production and CYP2A6 enzyme activity over time in the two types of silk/collagen scaffolds with small pores (73 ± 25 μm). (B) PHH albumin production and CYP2A6 activity over time in ‘collagen incorporated silk + autoclaved collagen’ scaffolds with either small or large pores (235 ± 84 μm). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.

Fig. 4. Functions of PHH-only and PHH + 3T3-J2 fibroblast co-cultures within silk/collagen scaffolds. ‘Collagen incorporated silk + autoclaved collagen’ scaffolds with small pores (73 ± 25 μm) were fabricated as described in methods. A suspension of either PHH alone (500k cells per scaffold) or PHH + growth-competent 3T3-J2 fibroblasts (1 : 1 ratio, 500k cells for each type) was seeded into the scaffolds. Albumin production and CYP2A6 enzyme activity over time are shown. *p ≤ 0.05, ***p ≤ 0.001, and ****p ≤ 0.0001.

Fig. 5. Functions of PHHs co-cultured with either growth-competent or growth-arrested 3T3-J2 fibroblasts in silk/collagen scaffolds of different pore sizes. ‘Collagen incorporated silk + autoclaved collagen’ scaffolds with either (A) small (73 ± 25 μm) or (B) large (235 ± 84 μm) pores were fabricated as described in methods. Albumin production and CYP2A6 activity are shown for the different culture models. *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001.

Fig. 6. Functions of PHH/fibroblast co-cultures in silk/collagen scaffolds, polymerized Matrigel only, and silk/collagen that contained polymerized Matrigel. ‘Collagen incorporated silk + autoclaved collagen’ scaffolds with (A) small (73 ± 25 μm) pores or (B) large (235 ± 84 μm) pores were fabricated as described in methods. The PHH + 3T3-J2 (growth-arrested, 1 : 1 ratio) co-cultures were either suspended in culture medium or a 4 mg mL⁻¹ solution of cold Matrigel (growth factor reduced). Next, the co-cultures in culture medium or in Matrigel were dispensed into the silk scaffolds as described in methods and referred to as ‘Silk’ and ‘Silk + Matrigel’ conditions, respectively. Additionally, PHHs in Matrigel was dispensed directly into the wells of a 48-well plate as a control condition (‘Matrigel’). Upon incubation at 37 °C, the Matrigel polymerized, thereby encapsulating the co-cultures. Albumin production and CYP2A6 activity are shown over time for the different culture models. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001 for ‘Silk + Matrigel’ vs. ‘Silk’. ⁺p ≤ 0.05, ⁺⁺p ≤ 0.01, ⁺⁺⁺p ≤ 0.001, and ⁺⁺⁺⁺p ≤ 0.0001 for ‘Silk + Matrigel’ vs. ‘Matrigel’.