Heat-Inactivation of Fetal and Newborn Sera Did Not Impair the Expansion and Scaffold Engineering Potentials of Fibroblasts

Abstract

Heat inactivation of bovine sera is routinely performed in cell culture laboratories. Nevertheless, it remains debatable whether it is still necessary due to the improvement of the production process of bovine sera. Do the benefits balance the loss of many proteins, such as hormones and growth factors, that are very useful for cell culture? This is even truer in the case of tissue engineering, the processes of which is often very demanding. This balance is examined here, from nine populations of fibroblasts originating from three different organs, by comparing the capacity of adhesion and proliferation of cells, their metabolism, and the capacity to produce the stroma; their histological appearance, thickness, and mechanical properties were also evaluated. Overall, serum inactivation does not appear to provide a significant benefit.

Keywords: 2D cell culture; heat-inactivation; mechanical properties; metabolism; serum; tissue engineering.

Figure 1

Adhesion and proliferation of fibroblasts primary populations depending on serum type and heat-inactivation status. DF is for fibroblast populations derived from skin dermis, BF is for fibroblast populations derived from bladder lamina propria, and VF is for fibroblast populations derived from vaginal stromae. HI- is for heat-inactivated, U- is for untreated, FBS is for fetal bovine serum, and NBCS is for newborn calf serum. Asterisks (*) illustrate significant differences. Two asterisks illustrate a p-value between 0.01 and 0.005. (A) Adhesion of fibroblast cell populations on a plastic Petri dish. The bars represent the mean ± standard deviation of the cell count 24 h after seeding normalized to the cell count obtained in HI-FBS. (B) Doubling time of fibroblast cell population calculated over a 3-day period. The bars represent the mean ± standard deviation of the doubling time of the cell population calculated from a growth curve established over 3 days. N = 3 independent fibroblast populations for each organ, n = 3 replicates.

Figure 2

Mitochondrial respiration of fibroblasts’ primary populations depending on serum type and heat-inactivation status. DF is for fibroblast populations derived from skin dermis, BF is for fibroblast populations derived from bladder lamina propria, and VF is for fibroblast populations derived from vaginal stromae. HI- is for heat-inactivated, U- is for untreated, FBS us for fetal bovine serum, and NBCS is for newborn calf serum. Basal mitochondrial activity (A) and maximal mitochondrial activity (B) were measured by a real-time extracellular flux analyzer. The bars represent the mean ± standard deviation of oxygen consumption rate (OCR). N = 3 independent fibroblast populations for each organ, n = 4 replicates.

Figure 3

Glycolytic activity of fibroblasts primary populations depending on serum type and heat-inactivation status. DF is for fibroblast populations derived from skin dermis, BF is for fibroblast populations derived from bladder lamina propria, and VF is for fibroblast populations derived from vaginal stromae. HI- is for heat-inactivated, U- is for untreated, FBS is for fetal bovine serum, and NBCS is for newborn calf serum. Basal glycolytic activity (A) and maximal glycolytic activity (B) were measured by a real-time extracellular flux analyzer. The bars represent the mean ± standard deviation of the extracellular acidification rate (ECAR). N = 3 independent fibroblast populations for each organ, n = 4 replicates.

Figure 4

Histological aspect of reconstructed stromae by fibroblasts’ primary populations depending on serum type and heat-inactivation status stained with Masson’s Trichrome protocol. DF is for fibroblast populations derived from skin dermis, BF is for fibroblast populations derived from bladder lamina propria, and VF is for fibroblast populations derived from vaginal stromae. HI- is for heat-inactivated, U- is for untreated, FBS is for fetal bovine serum, and NBCS is for newborn calf serum. N = 3 independent fibroblast populations for each organ, n = 4 tissues (three pictures have been taken of tissue). Scale bars are 100 µm.

Figure 5

Stroma thickness of reconstructed stromae by fibroblasts primary populations depending on serum type and heat-inactivation status. DF is for fibroblast populations derived from skin dermis, BF is for fibroblast populations derived from bladder lamina propria, and VF is for fibroblast populations derived from vaginal stromae. HI- is for heat-inactivated, U- is for untreated, FBS is for fetal bovine serum, and NBCS is for newborn calf serum. The bars represent the mean ± standard deviation of measurements on a photograph of the tissue slices stained with Masson’s Trichrome protocol. N = 3 independent fibroblast populations for each organ, n = 3 tissues (three pictures have been taken of tissue).

Figure 6

Maximal strength and elastic modulus of reconstructed stromae by fibroblasts primary populations depending on serum type and heat-inactivation status. DF is for fibroblast populations derived from skin dermis, BF is for fibroblast populations derived from bladder lamina propria, and VF is for fibroblast populations derived from vaginal stromae. HI- is for heat-inactivated, U- is for untreated, FBS is for fetal bovine serum, and NBCS is for newborn calf serum. Asterisks (*) illustrate significant differences. One asterisk is for p-value between 0.05 and 0.01; two asterisks is for p-value between 0.01 and 0.005. (A) Maximal strength of reconstructed stromae. The bars represent the mean ± standard deviation of the maximal strength in Newton (N) of the tissues using a uniaxial tensile test. N = 3 independent fibroblast populations for each organ, n = 4 tissues. (B) Elastic modulus of reconstructed stromae. The bars represent the mean ± standard deviation of the elastic modulus in MegaPascal (MPa) of the tissues using a uniaxial tensile test. The elastic modulus is in inverse relation to elasticity. N = 3 independent fibroblast populations for each organ, n = 4 tissues.

Figure 7

Ultimate tensile strength and failure strain of reconstructed stromae by fibroblasts primary populations depend on serum type and heat-inactivation status. DF is for fibroblast populations derived from skin dermis, BF is for fibroblast populations derived from bladder lamina propria, and VF is for fibroblast populations derived from vaginal stromae. HI- is for heat-inactivated, U- is for untreated, FBS is for fetal bovine serum, and NBCS is for newborn calf serum. Asterisks (*) illustrate significant differences. One asterisk is for a p-value between 0.05 and 0.01. (A) Ultimate tensile strength of reconstructed stromae. The bars represent the mean ± standard deviation of measurements of the UTS in MegaPascal (MPa) using a uniaxial tensile test. N = 3 independent fibroblast populations for each organ, n = 4 tissues. (B) Failure strain of reconstructed stromae. The bars represent the mean ± standard deviation of measurements of the failure strain in the percentage of the tissue length (%) using a uniaxial tensile test. N = 3 independent fibroblast populations for each organ, n = 4 tissues.