Cellmatrix type 1 p

The human myoblast cell line Hu5/E18 was cultured in collagen type I-coated 10-cm dishes (Iwaki, Japan) or Cellmatrix type I-P (Nitta gelatin, Japan) -coated dishes in myoblast medium containing DMEM, 20 % FBS, 2 mM L-glutamine, 0.5 % penicillin/streptomycin and 2 % Ultroser G serum substitute (Pall, France). For in vitro neuro-muscular co-culture, Hu5/E18 cells were plated at a density of 1×10³ cells/ml on a Cellmatrix type I-P-coated micro cover glass (Matsunami, Japan) and cultured until the cells reached approximately 50 % confluence. Then, the medium was changed to MNM, and the cells were cultured for 4 additional days, until the formation of myotubes. Partially dissociated EBs derived from KhES1 cells were plated onto Hu5/E18-derived myotubes at a density of 2.5×10²–5×10³ cells/cm² and co-cultured with the myotubes for 3–4 days in MNM.

The PC12 cells from the stock culture were suspended in the medium and plated at 4.0 × 10³ cells/90 μL/well (for the evaluation in the presence of Bt₂cAMP) or 2.0 × 10³ cells/90 μL/well (for the evaluation in the presence of NGF) in 96-well plates (Thermo Fisher Scientific K.K., Tokyo, Japan) with Cell-matrix type I–P (collagen) (Nitta Gelatin Inc., Osaka, Japan) and incubated in a humidified atmosphere of 5% CO₂ at 37 °C. After 24 h, 5 μL of Bt₂cAMP at 10 mM (final concentration: 0.5 mM) or NGF at 200 ng/mL (final concentration: 10 ng/mL), and 5 μL of each sample or the medium only (control) were added to the culture medium (the final concentration of each sample is indicated in the figures). At 24 h after the addition of Bt₂cAMP and the samples or at 48 h after the addition of NGF and the samples, the medium was aspirated, and the PC12 cells were fixed with a phosphate buffer (pH 7.2, 100 mM) containing 1% glutaraldehyde and stained with a Giemsa stain solution. Then the 96-well plates were washed twice with Milli-Q grade water. The number of cells bearing neurites longer than twice the diameter of one cell body after treatment was divided by the total number of cells, which was 300–400 cells per well.

HeLa (CCL-2, ATCC) and HEK293T (CRL-11268, ATCC) cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (D5796, Sigma) with 10% fetal bovine serum (Gibco) and 1% penicillin–streptomycin (Fujifilm Wako Pure Chemical Corporation) in a 5% CO₂ humidified incubator at 37 °C.
Immortalized human myogenic cells (Hu5/KD3)^{57 (link),58 (link)} were kindly provided by Dr. Naohiro Hashimoto (National Center for Geriatrics and Gerontology, Japan). Hu5/KD3 cells were cultured in primary myocyte growth medium (pmGM), which consists of high-glucose DMEM containing 20% FBS, 2% Ultroser G serum substitute (PALL), and 1% penicillin–streptomycin, in a 5% CO₂ humidified incubator at 37 °C. Culture dishes were manually coated with collagen using Cellmatrix Type I-P (Nitta Gelatin).

Poly(lactic-co-glycolic acid) (PLGA, the lactic acid/glycolic acid molar ratio = 75/25, molecular weight = 20,000 Da), thrombin, and paclitaxel (PTX) were purchased from Wako Pure Chemicals Industries, Ltd., Osaka, Japan. Poly(vinyl alcohol) (PVA, degree of polymerization = 1000, degree of saponification = 86–90%) was kindly supplied from Japan Vam & Poval Co., Ltd., Osaka, Japan. Gelatins with isoelectric points of 5.0 (pI5) and 9.0 (pI9) and the weight-averaged molecular weight of 100,000 Da were kindly supplied from Nitta Gelatin Inc., Osaka, Japan. Cellmatrix^® type I-P was purchased from Nitta Gelatin Inc., Osaka, Japan. Fibrinogen and adenosine 5′-diphosphate (ADP) were purchased from Sigma-Aldrich Inc., St. Louis, MO, USA. Bovine serum albumin (BSA) was purchased from Nacalai Tesque Inc., Kyoto, Japan. Coumarin-6 (CMR) was purchased from Tokyo Chemical Industry Co., Ltd., Tokyo, Japan. The reagents were used without further purification.

We performed a rapid two-step method^{61 (link)} to isolate mouse primary hepatocytes. We anesthetized the mice with pentobarbital sodium (50 mg/kg body weight) and perfused their liver tissue with prewarmed Hank’s balanced salt solution supplemented with 0.5 mM ethylene glycol tetraaceticacid (EGTA) and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) for 5 min, followed by digestion buffer (1000 mg/L of low-glucose Dulbecco’s modified Eagle’s medium [DMEM] supplemented with 0.8 mg/mL of collagenase type 1; Worthington Biochemical Corporation, Lakewood, NJ, USA). Next, we filtered single-cell suspensions through a 70-μm cell strainer (BD Falcon, Bedford, MA, USA) and centrifuged them at 50 × g for 1 min and collected the cell supernatants separately for the preparation of nonparenchymal cells, as described later. We washed the hepatocytes in the pellet twice, suspended them in 4500 mg/L of high-glucose DMEM supplemented with 1 μM insulin, 2 mM L-glutamine, 10 IU/mL of penicillin, 10 IU/mL of streptomycin, and 10% fetal bovine serum, and seeded them on 6-well plastic plates coated with collagen type I (Cellmatrix type I-P, Nitta Gelatin, Osaka, Japan) at a density of 300,000 cells/well. After 4 h incubation in a 5% CO₂ incubator at 37 °C, we changed the culture medium to discard floating unattached cells, and the growth medium was changed daily.