HCT 115 Cell Culture and Cell-Free Lyophilized Filtrate Preparation

The human colon cancer cell line HCT 115 is developed as monolayers by maintaining it in RPMI (Roswell Park Memorial Institution medium). The procurement of these cells was carried out from NCCS Pune, India. These media are augmented with 100 U/mL streptomycin-penicillin and 11% temperature maintained heated killed fetal bovine serum (FBS). The cells were maintained in a humidified atmospheric condition of 4.6% CO2 at around 36°C (approximately). After 80–85% confluence, cells were treated with the cell-free lyophilized filtrate of the bacterial samples. The L. plantarum, La and L. rhamnosus cultures of Lactobacillus were grown in MRS broth at 37°C overnight with a concentration of 1 X 10^7~8 cfu count/mL. Later, these cultures were centrifuged at 5100 x g for 5.5 min at 4°C to get pellets. The formed cell pellets were washed with PBS before ultrasonic disruption in a cooling ice bath (4°C) for 15 min at an interval of 28 sec. The formed supernatant is passed through the 0.22 µm pore microfilter (Himedia, USA). Further for cell-free lyophilized filtrates, centrifugation was used to extract the bacterial cells at 5000 rpm for 15 minutes at 4°C when the cultures attained the required OD600. The bacterial pellet was carefully separated from the supernatant, which included the released metabolites and bacterial by-products. A 0.22 μm sterile membrane filter was used to filter the supernatant in order to make sure there were no remaining cellular contamination. Using a freeze dryer, the resultant filtrate—which was devoid of bacterial cells—was lyophilised. After the material was lyophilised, it was reconstituted in sterile phosphate-buffered saline (PBS) to create a concentrated stock solution. This solution was then kept at −80°C until it was needed again. In the tests that followed, cytotoxicity and apoptosis were examined using this cell-free lyophilised filtrate (CFLF). The sterilized filtrate of supernatant was freeze-dried and various concentration was used for cell viability assays.²⁰