Rapid production of engineered human primary hepatocyte/fibroblast sheets

This data article contains data related to the research article entitled “Vascularized subcutaneous human liver tissue from engineered hepatocyte/fibroblast sheets in mice,” published in Biomaterials[1]. Engineered hepatocyte/fibroblast sheets (EHFSs) are used for construction of vascularized subcutaneous liver tissue without a pre-transplant vascularization procedures. Here, we described a rapid production technique of EHFSs by controlling fibroblast density and coating fetal bovine serum (FBS) onto temperature-responsive culture dishes (TRCDs). The human fibroblast monolayer formed on FBS-coated TRCDs within 1 h when seeded at a high density (at least 1.56×105 cells/cm2). The most rapid EHFS production was achieved soon after the adhesion of human primary hepatocytes onto the fibroblast layer.


a b s t r a c t
This data article contains data related to the research article entitled "Vascularized subcutaneous human liver tissue from engineered hepatocyte/fibroblast sheets in mice," published in Biomaterials [1]. Engineered hepatocyte/fibroblast sheets (EHFSs) are used for construction of vascularized subcutaneous liver tissue without a pre-transplant vascularization procedures. Here, we described a rapid production technique of EHFSs by controlling fibroblast density and coating fetal bovine serum (FBS) onto temperature-responsive culture dishes (TRCDs). The human fibroblast monolayer formed on FBS-coated TRCDs within 1 h when seeded at a high density (at least 1.56 Â 10 5 cells/cm 2 ). The most rapid EHFS production was achieved soon after the adhesion of human primary hepatocytes onto the fibroblast layer. & 2015 Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Rapid production of EHFSs was achieved approximately 3 h after the first inoculation of TIG-118 cells.

Fibroblast monolayer preparation by controlling cell density and FBS-coating to TRCD
Human fibroblasts (TIG-118 cells) formed a confluent monolayer within 1 h after inoculation with at least 1.56 Â 10 5 cells/cm 2 onto FBS-coated TRCDs ( Fig. 1A and B). Fibroblasts seeded at a lower density (1.04 Â 10 5 cells/cm 2 ) did not form confluent monolayers. Fibroblasts on uncoated TRCDs were unable to reach confluence despite high-density inoculation and showed non-uniform cell distributions ( Fig. 1C and D).

Human primary hepatocyte density for healthy culture on a FBS-coated TRCD
Human primary hepatocytes on FBS-coated TRCDs were not confluent within 1 day after inoculation under two conditions of hepatocyte densities (1.04 and 2.08 Â 10 5 cells/cm 2 ) (Fig. 2). After 3 days of culture, the hepatocytes showed a confluent monolayer. Hepatocytes at lower density (1.04 Â 10 5 cells/cm 2 ) were suitable for healthy culture because little dead cells were observed.

Effects of layer-by-layer procedure for stable, rapid production of EHFSs
Human primary hepatocytes adhered onto the confluent monolayer of fibroblasts for at least 2 h after hepatocyte inoculation. EHFSs were harvested from FBS-coated TRCDs soon after the adhesion of  hepatocytes by reducing the culture temperature from 37°C to 20°C for several minutes (Fig. 3A). Cosuspensions of hepatocytes and fibroblasts formed EHFSs, although the EHFSs were often selfdetached from FBS-coated TRCDs without temperature reduction before formation of continuous cell sheet format (Fig. 3B).

Cell preparation
Human primary hepatocytes were isolated from human liver tissues by perfusing collagenase (130 U/mL, Wako Pure Chemical, Osaka, Japan) [1]. Suspensions with 480% viable cells were used for this study. Normal human diploid fibroblast TIG-118 cells were purchased from Health Science Research Resources (JCRB0535; Osaka, Japan) [1,2].

Fibroblast monolayer preparation
To determine the proper conditions for the formation of a confluent monolayer, human fibroblasts were inoculated at 1.04, 1.56, or 2.08 Â 10 5 cells/cm 2 onto FBS-coated (2 h) or uncoated TRCDs. Minimum Essential Media supplemented with 10% FBS, 2 mM Lglutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin was used for fibroblast culture (all from Invitrogen, Carlsbad, CA).
At 2 h of culture, the confluency of fibroblasts was measured from phase-contrast micrographs using Win ROOF Version 6.3.0 (Mitani Corp, Fukui, Japan). Data are presented as mean 7standard deviation from 2 independent cell preparations.

Evaluation of human primary hepatocyte density
To evaluate the better density for human primary hepatocyte culture, hepatocytes were inoculated at 1.04 or 2.08 Â 10 5 cells/cm 2 onto FBS-coated TRCDs. Hepato-STIM Culture Medium (BD Biosciences, San Jose, CA) supplemented with 10% FBS, 2 mM L-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin was used for hepatocyte culture.