It has been shown previously that hyperthermia and HC may negatively impact the development of the embryo. These negative factors may act independently or interact together. According to the resulting effect, four types of interactions are recognized: nil effect (the lack of any observable interaction when two factors are used together), interference (protective), additive, and potentiation (Runner 1967). In our study, the cultures of fibroblasts were treated with HC added to the culture medium and cultured in vitro under different conditions to test the effect of elevated temperature and HC, as well as the effect of their interaction on cell proliferation.

Our results obtained using different approaches showed that a HC concentration of 0.1 mg/ml in culture media had a stimulating effect on the fibroblast proliferation. The positive effect of HC on cell proliferation was also proven by Arpels et al. (1964). They found that HC addition improved the population growth of several cell lines in a cell culture (lines HEp 1 and RPMI-41). HC at a concentration of 0.0025–0.01 mg/ml substantially extended the length of good growth, indicating that the cultures remained in excellent condition without refeeding the cultures with fresh medium for a longer time than all control cultures (Arpels et al.1964). Prolonged lifespan, shortened generation time, and higher plating efficiency were observed after the addition of cortisone at a concentration of 0.0025 mg/ml to culture medium (Macieira-Coelho 1966). Interestingly, the results of the two studies mentioned above showed a positive effect of HC addition on cell population growth at HC concentrations lower than one order in magnitude compared with our results. These very low concentrations seem to have an impact on cell proliferation over a longer period of time. However, an inhibitory effect of HC on the growth of fibroblasts obtained from cardiac explants has been reported. Inhibition of growth was demonstrated at HC concentrations as low as 0.2 mg/ml (Grossfeld and Ragan 1954). A similar effect of HC addition was also described in adult mouse, rabbit, and chicken fibroblasts that were exposed to cortisone at concentrations of 0.01–0.1 mg/ml. These concentrations had a significant inhibitory effect on cell population growth rate. Surprisingly, these concentrations of cortisone had no effect on the growth of embryonic fibroblasts, nor on the growth of adult human epithelial cells in vitro (Geiger et al. 1956). No effect of HC at a concentration of 0.2 mg/ml on cell population growth was observed in gastric and intestinal epithelial cells (Grossfeld and Ragan 1954). However, a negative effect of HC at concentrations of 0.01 mg, 0.1 mg, and 1 mg in vitro was proven using human cell lines, specifically three epithelial cell lines and one cell line derived from the connective tissue. In general, the epithelial cell lines required longer exposure to HC in contrast to cell lines derived from normal infant’s foreskin before any cytotoxic effect was detected (Kline et al. 1957).

The negative impact of HC on cell proliferation proven by studies mentioned above was caused by HC at concentrations ranging from 0.01 mg/ml to 1 mg/ml. However, our data showed that HC’s effect on proliferation is concentration specific. In our experiments, HC at a concentration of 1 mg/ml had an inhibitory effect on cell proliferation in contrast to HC at a concentration of 0.1 mg/ml, which had a stimulating effect on V79-4 cell proliferation. It seems that the effect could also be cell population (cell line) dependent. The studies of Grossfeld and Ragan (1954), Geiger et al. (1956), and Kline et al. (1957) also showed the inhibition of population growth at a concentration of 0.2 mg/ml and lower, which was proliferation-stimulating in our case. The differences could also be related to different initial cell numbers in the culture, different duration of the experimental manipulation of cell cultures, different culture conditions, or different effects of HC during short- and long-term experiments.

Interestingly, it has been shown that glucocorticoids (including HC) also evince cytotoxic effect (Guichard et al. 2015) and antiproliferative properties (Hammer et al. 2004).

It has been proven that glucocorticoids trigger cell death in certain cell types, such as monocytes (Schmidt et al. 1999), osteoblasts, and osteocytes (O’Brien et al. 2004) or tumor cells (Yamaguchi et al. 1999). The ability of glucocorticoids to inhibit cell growth has been used for decades in the treatment of childhood acute lymphoblastic leukemia (Gaynon and Lustig 1995).

Our results indicated that the effect of HC on the number of cells in the cell culture in our experiments was positive, even if not significant in all cases. In these cases, the increased cell death could be one of the factors playing a role in the final impact on cell culture growth since it has been shown that HC can induce apoptosis in distinct cell types, such as monocytes (Schmidt et al. 1999), tumor cells (Ikramova 2020), or keratinocytes (Guichard et al. 2015). However, the effect of HC on apoptosis seems to also be related to the cell line, growth of cells in suspension or monolayer, duration of HC treatment, and the HC dose used, and similarly to the effect of HC addition on cell proliferation.

Temperature has been found to have a great impact on biological processes in general. Cell cultures in vitro are valuable systems ideal for testing its impacts since the temperature can be controlled relatively easily.

Our results showed that long-term exposure to a temperature of 39 °C had a negative impact on fibroblast proliferation (Figs. 4, 5). However, many studies documented that short-term exposure to mild heat stress can positively regulate cell proliferation and viability. The mild heat stress with optimal temperatures between 39 °C and 40 °C supported proliferation and osteogenic differentiation of dental follicle stem cells (DFSCs; Rezai Rad et al. 2013). The periodic mild exposure of mesenchymal stem cells (MSCs) to the temperature of 41 °C was beneficial for cell viability, proliferation, and differentiation, and it led to the delayed senescence of the cells (Choudhery et al. 2015). Increased proliferation and enhanced neuronal proliferation after mild heat exposure was observed in the neural stem/progenitor cells (NSCs/NPCs) exposed to the temperature of 38.5 °C for 4 days (Hossain et al. 2017). Heat stress of 41 °C significantly stimulated viability of broiler fibroblasts within a short exposure time (24 h) unlike long-term exposure (for 3 days), which caused cell cycle arrest, induced apoptosis, and reduced cell viability (Siddiqui et al. 2020). Similar results were shown for chicken embryonic fibroblasts (CEFs) that were cultured at different temperatures (37 °C and 40–44 °C) for 6, 12, and 24 h, respectively. CEFs cultured at 41 °C presented significantly higher cell viability at distinct time points compared with the control. However, the cell proliferation and viability at temperatures above 41 °C were decreased in a time-dependent manner (Ibtisham et al. 2018). Results of these abovementioned studies demonstrated the beneficial effect of mild heat exposure for a short time on proliferation and viability of the cells. These findings are in accordance with our experiments revealing the positive effect of elevated temperature within first 48 h of incubation in case of doubling time evaluation. However, our data based on exposure for longer than 48 h showed a negative impact of elevated temperature on cell proliferation (colony formation assay and MIs). The negative impact of a long-term heat exposure has been shown to cause decreased cell proliferation and viability (Ibtisham et al. 2018; Siddiqui et al. 2020). The negative effect of high temperature on cell proliferation was shown in cardiac muscle tissue fragments of chick embryos that were exposed to temperatures of 5 °C, 12 °C, 20 °C, 30 °C, 39 °C, and 45 °C in vitro. It was observed that growth rate was highest at 39 °C and lowest at 45 °C. The tissue fragments kept at temperatures of 5 °C, 12 °C, and 20 °C did not multiply at all (Nemoto 1929). The prolonged exposure to supranormal temperatures of 42 °C and 44 °C on the growth of chick osteoblast in vitro did not show any lethal effect. However, the temperatures higher than 44 °C suppressed the cell population growth, and the death of cultures was observed after 105 min exposure to 47 °C, after 6 min exposure to 50 °C, and after 3.5 min exposure to 52 °C (Pincus and Fischer 1931). Interestingly, the inhibitory effect of supranormal temperatures in the range of 40–43 °C has been widely used to prevent tumor cells from proliferation during cancer treatment (Field and Bleehen 1979; Wust et al. 2002).

Based on these controversial observations, the effect of the elevated temperature is quite ambiguous. The response to heat stress may depend on cell type/line, developmental stage, temperature dose, and exposure time, similarly to the HC effect. The differences in the heat sensitivity of various cell lines were described. These differences could be related to the inherent variances between cell lines, growth of cells in a suspension or in a monolayer, observation of cells in vivo or in vitro, the tumorigenicity of cell lines, or the stage of the cell cycle and growth phase. It was shown that Chinese hamster ovary cells in late S-phase were more sensitive to heat stress than cells in early S-phase and in mitosis. Cells in G1 or G2-phases were the least sensitive to heat (Bhuyan et al. 1977). Similar results have been shown in S-phase and in mitotic cells, which were revealed as the most sensitive cell cycle phases to a heat shock of 43.5–46.5 °C. The cells in mitosis exposed to the heat shock failed to finish cytokinesis and became tetraploid (Westra and Dewey 1971). It was also proven that the cells grown in a suspension were slightly less sensitive to a temperature of 43 °C than the cells grown in a monolayer (Bhuyan et al. 1977).

Even though there are many variables that influence the effect of elevated temperature on cell proliferation and viability, it was shown that, although apoptosis and cell cycle arrest have been demonstrated in numerous studies, fever-range elevation of temperature or mild heat stress may positively regulate proliferation and differentiation of the cells via modulation of physical properties and activities of several regulatory proteins (Park et al. 2005). Interestingly, in pregnant women, basal body temperature (BBT) has been shown to remain elevated throughout approximately the first 4 months of a pregnancy (Zuck 1938; Buxton and Atkinson 1948; Siegler 1955). This slightly increased BBT could positively influence cell proliferation, which is one of the embryogenetic processes running during the first trimester of pregnancy.

Our results showed that HC at a concentration of 0.1 mg/ml exhibited a stimulating effect on cell proliferation. According to Runner’s (1967) four types of responses to concomitantly administrated substances, we could postulate that the main type of interaction between HC and elevated temperature during long-term exposure was protective (interference). Protective interaction means that the effect of one substance reduces the effect of another substance. In our case, the situation was slightly different because, unlike Runner (1967), one teratogen was of chemical and one of physical nature.

A deficit of cells in the brain has been observed in newborn guinea pigs prenatally exposed to short-term hyperthermia of 42.0–42.5 °C for 1 h on day 21 of gestation. The deficit of brain cells was related to increased cell death and to a delay in mitosis (Edwards et al. 1974).

Interestingly, in our study, the negative impact of hyperthermia on cell proliferation seems to be partially compensated by the addition of HC to culture media. It has been proven that HC can stimulate DNA synthesis (Takigawa et al. 1988), mitosis in a combination with growth factors (Hoshi et al. 1982), and cellular growth and increase the lifespan (Macieira-Coelho 1966). In our study, fibroblasts were exposed to the temperature of 39 °C, which could cause a delay in mitosis resulting in decreased proliferation. With the addition of HC, the negative effect of elevated temperature could be suppressed by stimulating the proliferation of delayed mitotic cells. The protective interaction between elevated temperature and HC might be cell line, time, and dose specific, as has been proven for independent experiments either with HC or hyperthermia.

To conclude, our results indicate that the elevated temperature (39 °C) had an ambiguous effect. After long-term exposure, decreased fibroblast proliferation was observed. However, after short-term exposure, elevated temperature seemed to slightly enhance cell proliferation compared with the control (37 °C). The addition of HC at a concentration of 0.1 mg/ml increased proliferation of fibroblasts at both tested temperatures. Interestingly, the interaction of both factors, elevated temperature (39 °C) and the addition of HC (0.1 mg/ml), positively stimulated proliferation in comparison to fibroblasts cultured at 39 °C without HC addition after long-term exposure. This suggests a protective effect of HC on proliferation of cells at elevated temperature.