Collagenase 4

Liver mesenchymal cells were isolated from BALB/c mice (Shanghai Laboratory Animal Center, Chinese Academy of Sciences, Shanghai, China) of 12 weeks old using retrograde perfusion via the inferior vena cava (IVC) [35 (link)]. Briefly, mice were anesthetized and perfused with pre-warmed solutions, including: i) 100 ml HBSS buffer containing 19 mg EGTA (Sigma-Aldrich), 357 mg HEPES (Sigma-Aldrich), and 0.5% penicillin and streptomycin for 20 min, ii) 0.05% pronase E (Sigma) solution containing 100 ml HBSS, 357 mg HEPES and 50 mg pronase E for 7 min, and iii) 0.05% collagenase IV (Gibco) solution containing 100 ml HBSS, 357 mg HEPES, and 50 mg collagenase IV for 5 min. After the in situ digestion, liver tissues were carefully excised and minced thoroughly, and they were further digested with pre-warmed 0.025% pronase E and 0.025% collagenase IV in vitro for 15 min. Cell suspension was collected and purified by 60% and 30% percoll (Sigma-Aldrich) density gradient centrifugation [35 (link)]. The freshly isolated liver mesenchymal cells were identified by morphology and biochemical phenotypes.

iPSC lines, iPSC-F [27 (link)], and iPSC-EGFP (from SiDanSai Biotechnology, Shanghai, China) were used in the present study. Some of the cellular morphologies were presented from iPSC-EGFP for its GFP expression distinguishing iPSCs from the stromal cells. iPSC lines were grown on mitomycin-C-treated mouse embryonic fibroblasts (CF1) with human ESC media consisting of 80 % DMEM/F12 (Invitrogen), 20 % knockout serum replacement (KSR) (Invitrogen), 1 mM l-glutamine (Invitrogen), 1 % non-essential amino acids (NEAA) (Invitrogen), 0.1 mM β-mercaptoethanol (Invitrogen), and 4 ng/ml basic fibroblast growth factor (bFGF) (Invitrogen). Media were changed every day. Colonies were split 1:4 every 7 days with 1 mg/ml collagenase IV (Gibco).
For 3D hematopoietic induction systems, 80 % confluent iPSCs were washed in phosphate-buffered saline (PBS) free of Ca²⁺ and Mg²⁺, and incubated with 1 mg/ml collagenase IV for 10–15 min. The colonies were pipetted into small cell aggregates and transferred to a 15-ml conical tube. Cells were washed twice in PBS and prepared for 3D induction systems.

Mononuclear cells were isolated from the SI-Lp and C-Lp as previously described [24 (link), 25 (link)]. In more detail, after the removal of Peyer's patches, small intestines and colons were flushed with PBS, opened longitudinally, and predigested twice with 5 mM EDTA and 1 mM DTT for 20 min at 37°C. After removing epithelial cells and fat tissue, the intestines were cut into small pieces and incubated in HBSS containing 0.5 mg/mL Collagenase D (Roche), 1 mg/mL Dispase II (Roche), and 5 U/mL DNase I (Sigma) for 20 min at 37°C in a shaking incubator. The digested tissues were washed, resuspended in 5 mL of 40% Percoll (Sigma), and overlaid on 2.5 mL of 80% Percoll in a 15 mL Falcon tube. Percoll gradient separation was performed by centrifugation at 1000 g for 20 min at 20°C. The interface cells were collected and used as Lp lymphocytes. Splenocytes and lymph node cells were isolated by mechanical disruption of the tissue. An additional digestion step with 1 mg/mL Collagenase IV (Gibco) for 20 min at 37°C was performed to increase dendritic cell extraction. Pancreatic intraislet lymphocytes were isolated by 3-step digestion with 1 mg/mL Collagenase IV (Gibco) followed by extensive washing with HBSS containing 5% FBS.

Tumor specimens were gently minced into small pieces and digested with 3 ml PBS containing 1 mg/ml collagenase IV (17104019; Invitrogen, Carlsbad, CA, USA) for 30 min at 37 °C. Cell suspensions were filtered twice and centrifuged at 1500 rpm for 5 min. Cell precipitates were resuspended in PBS, and tumor cells and tumor-infiltrating lymphocytes (TILs) were collected.

E6.0–6.5 embryos were dissected in FHM medium (Millipore) and the epiblast was isolated as described52 (link). Briefly, the embryonic portions were collected separately and grown in CDM (F12 Nutrient Mix/IDMEM, 5 mg/ml BSA, 450 μM Monothioglycerol, 7 μg/ml Insulin (Sigma), 15 μg/ml Transferrin (Roche), 100 IU/ml Pen/Strep, 1% Chemically Defined Lipid (Life Technologies), 12 ng/ml bFGF (R&D Systems), 20 ng/ml Activin A (Peprotech), 10 μM ROCK Inhibitor (Y27632, Tocris) at 37 °C and 6% CO₂. 3–4 days after plating individual epiblast outgrowths were partially dissociated into smaller pieces using a scalpel and transferred into freshly gelatin/DMEM/15% FCS-coated wells. After one week EpiSC colonies became visible, they were picked individually and processed as above. From the second passage onward the colonies were separated by 1mg/ml Collagenase IV (Invitrogen) treatment for two minutes at RT. After washing they were transferred into new wells, with special care to maintain big cell clusters. The ROCK Inhibitor improved proper attachment of the colonies to the plates53 (link). After long-term passaging the established EpiSCs stabilized in a more homogenous cell population.

Glomeruli from mice were isolated as described [33 (link)]. Briefly, mice were sacrificed and perfused with Dynabead M-450 (00388551, Invitrogen). The kidneys were digested in collagenase IV (1 mg/ml, Invitrogen) and pressed through a 100-μm cell strainer (BD Falcon, Bedford, MA), and then glomeruli were gathered using a magnetic concentrator, and the remaining fluids were centrifuged for collecting the tubules.
For isolation of rat glomeruli [36 (link)], SD rats were sacrificed in a sterile environment. Kidneys were harvested, decapsulated, minced, and then smashed down with a plunger through three sieves sequentially (200-100-60 mesh opening sizes). After washing and centrifugation, the glomeruli were resuspended in RPMI1640 with 10% FBS medium and plated on noncoated six-well plates for treatment with lentivirus expressing B7-1 (Genechem, Shanghai, China).

To passage iPCS, cells were washed with PBS and incubated with collagenase (Collagenase IV, 1 mg/ml, 17104–019; Invitrogen) and dispase (1 mg/ml, 17105–041; Invitrogen) for 45 min. After that, colonies were collected in a falcon tube containing iPSC media and were allowed to sediment for 2 min. The supernatant, containing residual collagenase/dispase, was removed, and the colonies were washed once with iPSC medium. The colonies were allowed to sediment again, and the supernatant was removed. Finally, colonies were mechanically broken up and plated onto fresh MEF feeders. Cells were passaged every 5–7 d (depending on the confluence of the plates) at a desired ratio.