An improved method for primary culture of normal cervical epithelial cells and establishment of cell model in vitro with HPV‐16 E6 gene by lentivirus

Abstract

Normal human cervical epitheliums infected with HPVs gene in vitro are underlying molecular models to investigate physiological mechanisms of cervical epithelia and cervical disease. The current study aimed to establish a modified culture method for cervical epithelium and explore the feasibility of transfection with HPV-16 E6 gene mediated by lentivirus in primary cervical cells. The cells were dissociated enzymatically using Dispase II combined with 0.25% Trypsin-0.01% ethylenediamine tetracetic acid (EDTA) or Collagenase I. The detached effectiveness of Dispase II at different times was compared. Isolated cells were cultured and subcultured in modified keratinocyte serum-free medium (K-SFM) supplemented with 5% fetal bovine serum (FBS) or K-SFM alone. Cytokeratin was used as the identification of cervical epitheliums. Proliferative capacity and growth curve of cervical epitheliums were evaluated by cell counting kit-8 (CCK-8). The cells at passages 3 were used to infect with HPV-16 E6 gene by lentivirus. The expression of green fluorescent protein (GFP) presented in the infected cells was observed via fluorescence microscopy and the levels of E6 mRNA were detected by quantitative real-time PCR (qRT-PCR). The results indicate that cervical epithelial cells can be isolated successfully by Dispase II combined with 0.25% Trypsin-0.01% EDTA method for 20 hr and maintained for five or six passages in K-SFM medium with 5% FBS. The present study proposed a brief and high-yield protocol for isolation and culture of human cervical epitheliums. Moreover, an infected cell model with HPV-16 E6 gene mediated by lentivirus was established which can do duty for studies in vitro on the carcinogenic mechanism of HR-HPVs.

Isolation of Biopsy-Derived, Human Cervical Keratinocytes Propagated as Monolayer and Organoid Cultures

“…Successful culturing largely depended on the nature of the biopsy with regard to its size and digestion ability before plating. An interesting insight regarding the latter was recently published by Fan and colleagues 25 finding that enzymatic dissociation of keratinocytes worked better with dispase II combined with 0.25% trypsin–0.01% EDTA than with collagenase I. This may be due to the relative content of stromal components in cervical biopsies such as fibronectin versus collagen.…”

“…An inquiry, with one of them replying, revealed that they guarantee 3 passages with a total of 10 PDs and a PDT typically between 40 to 70 hours (depending on medium composition). In the Fan and colleagues’ study 25 , where keratinocyte cultures were likewise obtained from hysterectomies, 5–6 passages with 1 PD per passage was reported. Taken together, this suggests that our samples outperform both these accomplishments despite the fact that they originated from biopsies: an average of 8 passages, each with 3 PDs, and a PDT of 27 hours, expecting to yield several hundred million cells while also preparing cell stocks.…”

Establishment and optimization of epithelial cell cultures from human ectocervix, transformation zone, and endocervix optimization of epithelial cell cultures

“…Thus, there is a need to develop and optimize methods for growing cervical cells from each region. Although several methods have been used to culture cervical cells based on serum‐free medium or serum‐supplemented culture medium (Chapman, Liu, Meyers, Schlegel, & McBride, ; Fan et al, ; Fu, Quintero, & Baker, ; Rheinwald et al, ; Stanley & Parkinson, ), there has been no systematic evaluation of serum‐free culture media for cells from different cervical regions.…”

Experimental Approaches to Deriving Primary Tumor Cell Cultures From Cervical Cancer Biopsies: A Comparative Analysis of Published Data and Own Experience

“…применяли инкубацию с 0,14% коллагеназой I типа и 0,01% ДНКазой в среде RPMI-1640 при +37°С (в течение 2 ч) или +4°С (в течение ночи) во флаконах большого объема на магнитной мешалке (Santin, 2005). Для получения нормальных цервикальных эпителиоцитов возможно использование диспазы II типа в сочетании с 0.25% трипсином-0.01% EDTA в течение 20 ч (Fan, 2017). Ряд авторов также включают в процесс пробоподготовки фильтрацию полученной суспензии клеток через 150 мкм-нейлоновую сетку с целью получения единичных клеток для их дальнейшего равномерного посева на пластик с определенной плотностью (Santin, 2005;Magaldi, 2012), однако, другие исследователи указывают на отсутствие необходимости данного этапа (Liu, 2016).…”

“…Необходимость варьирования содержания сыворотки обусловлена тем, что при высокой ее концентрации (10%) может наблюдаться ускоренный рост фибробластов и дифференцировка («старение») цервикальных кератиноцитов, а в бессывороточной среде кератиноциты, извлеченные из ткани (нормальной или опухолевой), плохо прикрепляются к поверхности и погибают (Bononi, 2012;Liu, 2016). Поэтому рекомендуется посев кератиноцитов проводить при высокой концентрации фетальной сыворотки (5-10%), а после прикрепления клеток снижать ее содержание (до полного отсутствия) (Santin, 2005;Bononi, 2012;Liu, 2013Liu, , 2016Fan, 2017). Для культивирования клеток ЦИН, Bononi и соавт.…”