Collagenase type 1

Hindlimb skeletal muscles were minced and digested with a mixture of type I collagenase and dispase B (Roche Applied Science, Basel, Switzerland). The obtained cells were filtered, centrifuged, and cultured in growth medium (F-10 Ham's medium supplemented with 20% FBS, 4 ng/ml basic fibroblast growth factor, and 1% penicillin–streptomycin) on collagen-coated cell culture plates at 37 °C, 5% CO₂. The cell differentiation was induced in differentiation medium (DM) containing 2% HS and then cultured for 36 and 72 h, respectively.

The entire protocols were conducted in accordance with the Ethics Committee of the School of Stomatology, Wuhan University. The isolation of human dental pulp cells (hDPSCs) was performed as our previous study.^{46 (link)} Briefly, immediately after patients’ healthy third molars (aged 15–26) were extracted, the samples were transported to ice-cold phosphate-buffered saline (PBS, Hyclone, Logan, UT) with 100 U·mL^–1 penicillin/streptomycin (PS, Hyclone) and rinsed repeatedly. The dental pulp tissues from five individual patients were then softly removed, pooled together, cut into tiny pieces and digested by 3 mg·mL^–1 type I collagenase and 4 mg·mL^–1 Dispase II (Roche) for 1 h at 37 °C in water batch, aided by shaking every 10 min. After enzymatic dissociation, cell suspension was passed through a 40 μm Falcon Cell Strainers. The freshly obtained cell suspension was then centrifuged for 5 min at 1000 r·min^–1. The cell pellet was then resuspended into alpha Dulbecco’s modified Eagle’s medium (α-MEM, Hyclone, Logan, UT) with 20% fetal bovine serum (FBS, Gibco) and 100 U·mL^–1 PS, at 37 °C and 5% CO₂ in 25 cm² flasks. Cells started to expanded; and after 10 days, the cultured cells were trypsinized, rinsed and prepared for the single-cell suspension used for scRNA-seq or FACS cell sorting.

This study was conducted in accordance with the ethical standards laid out in the Declaration of Helsinki (1975) and was approved by the Institutional Ethics Committee at China Medical University. Adipose tissue samples were obtained with informed consent from patients at Shengjing Hospital. The ADSCs were isolated and harvested as previously described [24 (link)]. Briefly, adipose tissues were digested with type I collagenase (Roche Diagnostic, Mannheim, Germany) under gentle agitation at 37°C for 30 min. The enzyme activity was neutralized with FBS, and the suspensions were centrifuged at 300 g for 10 min to remove floating mature adipocytes. The cells were seeded into DMEM medium supplemented with 10% FBS. The cultures were maintained at 37°C in a 5% CO₂ incubator. After 48 h, non-adherent cells were removed, and adherent cells were washed 3 times with phosphate-buffered saline (PBS). The medium was changed every 3 days, and the cells were subcultured when the monolayer of adherent cells reached confluence. ADSCs were used in experiments between passages 3–6.

Three allanto-amniotic membranes were obtained at the term of a normal and physiological pregnancy, transported at 4 °C in phosphate-buffered saline solution (PBS) supplemented with 4 mg/mL amphotericin B (Euroclone), 100 U/mL penicillin (Euroclone) and 100 mg/mL streptomycin (Euroclone), and were processed within 8 h of collection. Amniotic membranes were mechanically separated from the overlaying allantois and cut into small sections of 9 cm² each, for a total weight of approximately 12 g and an extension of 630 cm². Then, amniotic fragments were incubated for 9 min at 38.5 °C in PBS containing 2.4 U/mL dispase (Becton Dickinson & Company, Milan, Italy). After a final incubation of 5–10 min at room temperature in HG-DMEM (high-glucose Dulbecco’s modified Eagle’s medium, Euroclone) supplemented with 10% fetal bovine serum (FBS) and 2 mM L-glutamine, fragments were digested with 1 mg/mL collagenase type I and 20 µg/mL DNase (Roche, Mannheim, Germany) for 3 h at 38.5 °C.
Fragments were then removed, and the products of enzymatic digestion were passed through a 100 µm filter. Dissociated cells were collected through centrifugation at 250× g for 10 min.
Isolated cells were defined as AMSCs.

Five different protocols for enzymatic digestion and cell isolation of cardiac tissue was assessed in murine and human heart tissue, based on previous protocols applied to mouse tissue (Farbehi et al., 2019 ; Gladka et al., 2018 (link)) and one which had been used on human tissue (Bajpai et al., 2018 (link)). Dissected tissue was placed in a tube and incubated with one of the following: (1) HBSS with 1 mg/ml Collagenase Type II (ThermoFisher Scientific, Waltham, MA, USA) and 3 mM CaCl₂, (2) HBSS with 0.5 mg/ml Elastase (Sigma Aldrich, St Louis, MO, USA) and 3 mM CaCl₂, (3) HBSS with 1 mg/ml Collagenase Type II and 0.5 mg/ml Elastase, (4) DMEM with 450 U/ml Collagenase Type I and 60 U/ml Hyaluronidase or (5) DMEM with Liberase (Roche, Basel, Switzerland). Tissue was digested for 2 h at 37 °C in protocol 1–3 and 1 h at 37 °C in protocol 4–5. Mouse tissue was digested for 20 min in all protocols. After incubation, cells were passed through 100 or 200 μM cell strainers, washed with HBSS and resuspended in medium for primary cells.

SCG were removed from adult Swiss Webster mice of either sex (postnatal day 56), cut in half, and treated for 20 min at 37°C with 20 U/mL papain (Worthington Biochemical, Cat# LS003126) in a calcium- and magnesium-free (CMF) Hank’s buffer (Gibco, Cat# 14170-112) containing 137 mM NaCl, 5.36 mM KCl, 0.33 mM Na₂HP₄, 0.44 mM KH₂PO₄, 4.2 mM NaHCO₃, 5.55 mM glucose, and 0.03 mM phenol red. The ganglia were then treated for 20 min at 37°C with 3 mg/mL collagenase (type I; Roche Diagnostics, Cat# 10103586001) and 4 mg/mL Dispase II (Roche Diagnostics, Cat# 37045800) in CMF Hank’s buffer. Cells were dispersed by trituration with a fire-polished glass Pasteur pipette in a solution composed of two media combined in a 1:1 ratio: Leibovitz’s L-15 medium (Gibco, Cat# 11415-064) supplemented with 5 mM HEPES and DMEM/F12 medium (Gibco, Cat# 11330-032) and plated onto coverslips. Then cells were incubated at 37°C (5% CO₂) for 2 hr, after which Neurobasal medium (Gibco, Cat# 10888-022) containing B-27 supplement (Gibco, Cat# A3582801), and penicillin and streptomycin (Sigma-Aldrich, Cat# P4333) was added to the dishes. Cells were stored at room temperature and used within 48 hr.