Functional 3D Human Primary Hepatocyte Spheroids Made by Co-Culturing Hepatocytes from Partial Hepatectomy Specimens and Human Adipose-Derived Stem Cells

<div><p>We have generated human hepatocyte spheroids with uniform size and shape by co-culturing 1∶1 mixtures of primary human hepatocytes (hHeps) from partial hepatectomy specimens and human adipose-derived stem cells (hADSCs) in concave microwells. The hADSCs in spheroids could compensate for the low viability and improve the functional maintenance of hHeps. Co-cultured spheroids aggregated and formed compact spheroidal shapes more rapidly, and with a significantly higher viability than mono-cultured spheroids. The liver-specific functions of co-cultured spheroids were greater, although they contained half the number of hepatocytes as mono-cultured spheroids. Albumin secretion by co-cultured spheroids was 10% higher on day 7, whereas urea secretion was similar, compared with mono-cultured spheroids. A quantitative cytochrome P450 assay showed that the enzymatic activity of co-cultured spheroids cultured for 9 days was 28% higher than that of mono-cultured spheroids. These effects may be due to the transdifferentiation potential and paracrine healing effects of hADSCs on hHeps. These co-cultured spheroids may be useful for creating artificial three-dimensional hepatic tissue constructs and for cell therapy with limited numbers of human hepatocytes.</p> </div

Co-cultured spheroids had higher viability and aggregated more rapidly than mono-cultured spheroids.

<p>Spheroidal formation and viability of cells in mono-cultured spheroids cultured for (A) 3 days, (B) 7 days and in co-cultured spheroids cultured for (C) 3 days and (D) 7 days in concave microwells. Scale bars, 500 µm, (E) Diameter analysis of mono-cultured spheroids and co-cultured spheroids in 500 µm concave microwells. Data represent the means ± standard deviations of 10 independent experiments (*p<0.0001, two-tailed test).</p

Directed Evolution of Human Heavy Chain Variable Domain (V_H) Using <i>In Vivo</i> Protein Fitness Filter

<div><p>Human immunoglobulin heavy chain variable domains (V_H) are promising scaffolds for antigen binding. However, V_H is an unstable and aggregation-prone protein, hindering its use for therapeutic purposes. To evolve the V_H domain, we performed <i>in vivo</i> protein solubility selection that linked antibiotic resistance to the protein folding quality control mechanism of the twin-arginine translocation pathway of <i>E. coli</i>. After screening a human germ-line V_H library, 95% of the V_H proteins obtained were identified as V_H3 family members; one V_H protein, MG2x1, stood out among separate clones expressing individual V_H variants. With further screening of combinatorial framework mutation library of MG2x1, we found a consistent bias toward substitution with tryptophan at the position of 50 and 58 in V_H. Comparison of the crystal structures of the V_H variants revealed that those substitutions with bulky side chain amino acids filled the cavity in the V_H interface between heavy and light chains of the Fab arrangement along with the increased number of hydrogen bonds, decreased solvation energy, and increased negative charge. Accordingly, the engineered V_H acquires an increased level of thermodynamic stability, reversible folding, and soluble expression. The library built with the V_H variant as a scaffold was qualified as most of V_H clones selected randomly were expressed as soluble form in <i>E. coli</i> regardless length of the combinatorial CDR. Furthermore, a non-aggregation feature of the selected V_H conferred a free of humoral response in mice, even when administered together with adjuvant. As a result, this selection provides an alternative directed evolution pathway for unstable proteins, which are distinct from conventional methods based on the phage display.</p></div

hADSCs in co-cultured spheroids exhibited hepatocyte-like features.

<p>(A) The expression of mRNA for the co-cultured spheroids on day 3, day 7, and hADSC mono-cultured spheroids on day3. (B) Quantification of the relative gene expressions to GAPDH. Data represent the means ± standard deviations of 3 independent experiments. (*p<0.1, **p<0.05, ***p<0.001, two-tailed test).</p

Co-cultured spheroids showed higher liver specific function and more stable metabolic function than mono-cultured spheroids.

<p>Analysis of metabolic function of mono-cultured spheroids and co-cultured spheroids, measured as secretion of (A) albumin and (B) urea. Data represent means ± standard deviations of eight independent experiments. Immunostaining for cytochrome P450 reductase (red) in (C) mono-cultured spheroids and (D) co-cultured spheroids cultured for 9 days. Nuclei were stained with DAPI (blue). Scale bars, 50 µm. (E) Luminescence assay of cytochrome P450 activity in mono-cultured and co-cultured spheroids. Cytochrome P450 3A enzyme activity were measured based on the degree of luminescence emitted by adding detection reagent and measured with a luminometer. Data represent means ± standard deviations of 3 independent experiments.</p

Far-UV CD spectra for the detection of reversible folding.

<p>(A) MG2x1. (B) MG8-6. (C) MG8-14. (D) Modified MG8-14 [L50W]. Black lines indicate profiles for V_H in native state at 25°C; red lines indicate the profile for V_H denatured at 85°C; and green lines indicate profiles for V_H renatured at 25°C.</p

Thermodynamic stability.

<p>(A) Representative V_H domains selected from the germ-line library. (B) Representative V_H domains selected from the MG2x1 frame-mutation library. The black bold line indicates the profile of the parental V_H, MG2x1, prior to mutation. Folding fraction was converted from the temperature-scouting CD profile at a fixed wavelength (230 nm).</p

Co-cultured spheroids showed healthy hepatocytic ultrastructure.

<p>Ultrastructural feature of the co-cultured spheroids by transmitting electron microscopy (TEM) on day 7 of culture. The spheroids are characterized by tight junctions (Tj) between adjacent cells, distinct nuclei (N), abundant mitochondria (M), peroxisomes (P), rough endoplasmic reticulum (rER), collagen accumulation (Col), glycogen vesicles (Gl), and bile canaliculi (B).</p

Only co-cultured model can form compact spheroidal shape.

<p>SEM images of mono-cultured spheroids cultured for (A) 3 days, (B) 9days and co-cultured spheroids cultured for (C) 3 days and (D) 9 days. Scale bars, 10 µm. Immunostaining for AE-2 (green) and actin (red) in (E) mono-cultured spheroids and (F) co-cultured spheroids cultured for 9 days. Scale bars, 50 µm. Scanning electron microscopy images of sectioned co-cultured spheroids cultured for (G) 3 days and (H) 9days. Inner structure of co-cultured spheroids is porous which involves good diffusion of nutrient and oxygen into the spheroid. Scale bars, 10 µm.</p

Schematics procedures of mono- and co- cultured spheroid formation.

<p>hHeps were isolated from the healthy resection margins of liver samples obtained during partial hepatectomy, and hADSCs were isolated from leftover human subcutaneous adipose tissue of patients undergoing plastic surgery or liposuction. Cells were seeded onto concave microwells and cultured for a few days to form cell aggregates and spheroids.</p