Enzyme‑injected method of enzymatic dispersion at low temperature is effective for isolation of smooth muscle cells from human esophagogastric junction

Abstract

The present study was conducted to examine the feasibility of in vitro isolation and primary culture of smooth muscle cells (SMCs) from the esophagogastric junction (EGJ).Smooth muscles of EGJ were harvested from 23 patients with esophageal cancer during esophagostomy from January 2015 to December 2017. Enzymatic dispersion (ED) was performed for isolation. Collagenase II and Trypsin/EDTA were applied by enzyme injection (EI) into tissue fragments or immersion of tissue fragments into enzyme solution. Growth characteristics and proliferation [Cell Counting Kit-8 (CCK-8)] of cells were recorded for both smooth muscle cell medium (SMCM) and DMEM/F12 containing 10% newborn bovine serum (10%-F12). All ED methods could isolate primary cells; EI was the most effective method with low collagenase II concentration (0.5 mg/ml) at 4˚C for 14-24 h. Primary cells demonstrated mainly spindle-and long-spindle-shaped with 'hills and valleys' morphology. The CCK-8 assay in SMCM showed better proliferation results than in 10%-F12. After passaging for 4-8 generations in SMCM or 2-4 generations in 10%-F12, cells enlarged gradually with passages and lost spindle structures. mRNA and proteins of α-smooth muscle actin (α-SMA), smooth muscle 22 α (SM22α), vimentin, desmin, CD90 and proliferating cell nuclear antigen were detected in tissues and cells with different levels of expression. SMCs of esophageal circular muscle, esophageal longitudinal muscle, gastric circular muscle near sling in gastric bottom and gastric circular muscle near clasp in lesser gastric curvature, all cultured in 10%-F12, exhibited superior smooth muscle phenotypes compared with SMCs cultured in SMCM in terms of α-SMA, SM22α and vimentin expression. The EI method of ED at low temperature appears effective for isolation and primary culture of SMCs from human EGJ in vitro.

The effects of acetylcholine on intracellular calcium fluorescence in smooth muscle cells of human esophagogastric junction cultured in vitro

“…ED and EC-T methods were applied to obtain primary cells from smooth muscle strips. Protocols of both methods were referred to previous studies, [15][16][17][18][19] but were improved to simple processes as described below. 15 Smooth muscle strips for the ED method were cut into 5-mm ×5-mm fragments and soaked in collagenase II-DMEM/F12 solutions at a concentration of 0.5 mg/ml (collagenase II ≥125 CDU/mg) (VETEC, Sigma-Aldrich, St. Louis, MO, USA).…”

“…Previous procedures were followed. 3,14,15 Sling, Clasp, abdominal esophageal circular muscle (ECM), and longitudinal muscle (ELM) samples were prepared in 5-15 mm ×5-10 mm strips. Samples were quickly placed into 1.5-ml Eppendorf tubes (EP) with 1 ml DMEM/ F12 (Thermo Fisher Scientific, Waltham, MA, USA) and 200 µl penicillin/streptomycin (P/S) (BI, Beit Haemek, Israel).…”

“…Primary culture and identification of human esophageal SMCs or myofibroblasts has been described previously. 13,[15][16][17] This study improved processes for SMC culture by using the enzyme solution-injected method for ED and residual tissues for EC-T.…”

“…The improved protocols are as follows. 15 First, appropriate enzyme solution (collagenase II-DMEM/F12 solutions, 0.5 mg/ml) injected into tissue fragments can increase the contact between tissue and enzyme solution. Second, digestion at low temperature (4℃) can reduce the damage of SMCs by enzyme solution.…”