In the present study, we discover a novel approach to cell culture. Firstly, tissue block was continuously subcultured for several passages to achieve cells; as the control, meanwhile, the cells from tissue block at passage 1st were traditionally passed to 10 passages. Then, cells at passages 1st, 5th and 10th under two culture methods were respectively collected. Lastly, alterations including cellular viability, proliferation, senescence and apoptosis were determined by a serial of cytoproteological techniques to compare the differences between two culture methods. Our findings reveal the superiority of CASTB method to traditional subculture one in FBs and a potential of supplying massive primary cells by the new culture approach.
Technology of cell culture is essential to for life science research. To get more cells, more superior vehicles for cell culture need to be developed. At present, several techniques are applied in primary cell culture, mainly tissue block culture method and enzymatic digestion method (Bass et al. 2011; Morino et al. 2019); but they have some imperfections, like low cell production, poor cellular vitality and materials wasting (Wang et al. 2012; Han et al. 2013; Romano et al. 2020; Srikanth et al. 2021). The current study, therefore, explored a newly improved method of cell culture, namely CASTB (continuous adherence subculture of tissue block). We herein chose dermal FBs as the target cells and cultured them with CASTB method, by which the tissues discarded in the traditional method could be continued to use effectively. Our findings showed that cells crawled out after tissue block first adherence for 2–3 days, entering the logarithmic growth period in 5–7 days and covering 90–95% of the bottle bottom in 9–10 days; they grew adherently with a spindle or polygon shape, specifically expressing vimentin marker, which was consistent with the previous reports (Kisiel et al. 2019; Zhang et al. 2019) and thus confirmed the cells as FBs. More importantly, cells by CASTB displayed few changes no matter how many passages the tissue block experienced. Even undergoing repeated digestion and passages, the tissue block still attached to the wall stably, and produced an increasing number of cells. This is good for making full use of tissue blocks and avoiding the waste of materials, especially some insufficient or scarce tissues.
In CASTB way, however, the production of cells might be influenced by several factors, especially enzyme digestion style and tissue block adhesion. The previous reports showed that the vitality of primary cells decreased with a poor adherent ability of tissue block after a 8-12h treatment with collagenase type II in the traditional way, the reason probably being related to the long-time digestion of collagenase (Chen et al. 2020; Jagadeeshaprasad et al. 2022). Consequently, mixed enzyme digestion of collagenase II and trypsin for 30 minutes was employed in the present study to treat tissue blocks. Our results revealed that a short-time mixed enzyme digestion not only benefited the attachment of tissue block, but also maximized the primary cells migration from tissue block. Meanwhile, it only took 2–3 days in CASTB way for cells migration from tissue block, being shorter than the 5–6 day in the traditional one. This mostly has something to do with the shorter digestion time in CASTB way and the less damage to cells.
On the other hand, the successful adhesion of tissue block was quite critical to cell culture, which involved a serial of elements, primarily including size, distribution density and drying time of tissue block. Previously, the tissue block was often cut into about 1 × 1mm2 in size with neat and smooth edges (Proudfoot and Shanahan 2011; Voicekhovskaya et al. 2012; Mutlu et al. 2020). Nevertheless, we, after many pre-experiments, found that 2×2mm2 size of tissue block was optimum because the too-small tissue block tended to shrink during the adherent process and be unfavorable to reuse, whereas the too-large one kept the cells poor in nutrients and easy to die. The present results showed that few alterations emerged from 2×2mm2 sized tissue block even undergoing 10 times passages; no difference appeared in the proliferation, senescence or apoptosis rate of FBs from tissue blocks at passages 1st, 5th and 10th. It indicated that proper-sized tissue blocks and cells from them exhibited a good survival state no matter in which passage. Secondly, the tissue blocks should distribute regularly when attaching to the wall. According to previous literatures (Naz et al. 2019), the tissue blocks were seeded at an interval of 0.5cm in our study, which greatly benefited cell-cell interactions and thereby reduced cell apoptosis (Hass et al. 2009; Otte et al. 2013; Hendijani et al. 2017). Besides, the length of drying time largely affected tissue block adhesion and cells survival. The short drying time favored cell survival over tissue block adherence, while the long one was the opposite (Mori et al. 2015); for this reason, the drying time was set at 2 hours in the current experiment, and our findings confirmed it as the most appropriate time. Therefore, it is suggested the optimal parameters of tissue block adhesion as size of 2×2mm2, interval of 0.5cm and drying time of 2 hours during the attachment of tissue blocks in CASTB way.
Now that the CASTB method exerted a good effect on cell culture especially primary cell culture, does it surpass the traditional one? To compare how well various culture techniques worked, the traditional subculture counterpart was chosen as the control. FBs from the tissue blocks that continuously went through 10 times were investigated in the current experiment, as well those with 10 conventional subcultures. We discovered that FBs by the CASTB way exhibited the same size, shape and growth state as those by the traditional one; no difference existed in the growth curve between FBs from the first culture of tissue block and those from the first subculture, indicating that the growth rate and culture cycle in both groups at passage 1st were consistent. With the increase of passage times, however, obvious changes occurred in the growth state, cellular status and proliferation rate of FBs by the traditional subculture; namely, cellular growth and proliferation rate gradually slowed down, and senescent cells grew dramatically with deformed/fragmented nuclei at 9–10 passages, which squared with the findings from Yingli Zhang et al., who found that the survival ability and proliferation capacity of FBs significantly decreased after the cells were passed over four generations (Chang et al. 2019). Conversely, above alterations scarcely appeared in FBs by CASTB regardless of passage 1st, 5th or 10th, revealing that cellular growth/proliferation conditions of FBs by the CASTB method excelled over those by the traditional one.
Further to analyze the specific differences of both culture methods on FBs, indicators about cellular proliferation activity, senescence and apoptosis were investigated. Our results displayed few difference appearing in cellular proliferation, senescence, apoptosis as well the expressions of Ki67, PCNA and Bcl-2 in FBs by CASTB; nevertheless, FBs by traditional subculture, with passages increasing, exhibited notable reductions in proliferation rate and Ki67/PCNA levels and apparent enhancements in senescence, apoptosis rate and Bcl-2 expression. It was suggested that FBs cultured by CASTB always remained stable activity, well growth and powerful proliferation capacity with low senescence and apoptosis; but those cultured by traditional subculture behaved an opposite trend, further manifesting a fine superiority of the CASTB method over the traditional subculture.