An Assay To Evaluate the Anticancer Effects of Lactobacillus Cell-Free Supernatant

Abstract

Source: Lee, J., et al. Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells. J. Vis. Exp. (2020).

This video demonstrates an in vitro assay to study the anticancer effects of Lactobacillus cell-free supernatant (LCFS). Upon exposing colorectal cancer spheroids to varying doses of LCFS, the interaction with the bacterial metabolites in LCFS induces apoptosis in the cancer cells, disrupting the spheroid structure and leading to cell death in a dose-dependent manner.

Protocol

1. Bacterial cell cultures and preparation of Lactobacillus cell-free supernatant (LCFS)

NOTE: Steps 1.2 – 1.9 are conducted in an anaerobic chamber.

1. Prepare a De Man–Rogosa–Sharpe (MRS) agar plate and broth containing L-cysteine and sterilize by autoclaving.
2. Pre-incubate the MRS agar plate in H₂ anaerobic chamber maintained at 37 ˚C with 20 ppm oxygen.
3. Thaw Lactobacillus bacterial stock and inoculate the agar plate with the bacterial culture (Figure 1A (i)).
4. Incubate bacteria for 2 – 3 days in H₂ anaerobic chamber at 37 ˚C and 20 ppm oxygen until single bacterial colonies are obtained.
5. Wash and dry the Hungate type anerobic culture tube. Autoclave the culture tube at 121 ˚C for 15 min.
6. Then incubate the tube in H₂ anaerobic chamber at 37 ˚C and 20 ppm oxygen to remove oxygen.
7. Place 2 – 3 mL of MRS broth into the tube. Seal the tube with a butyl rubber stopper and screw the cap.
8. Obtain a single colony with a loop and place it into the 1.5 mL culture tube with 500 µL of 1x PBS. (Figure 1A (ii)).
9. Suspend the colony using a 1 mL syringe (Figure 1A (iii)). Do this by, inserting the needle of the 1 mL syringe in the center of the tube lid, aspirating the suspended colony and then resuspending it back into the MRS broth media. (Figure 1A (iv)).
10. Incubate the MRS broth media in a shaker incubator for 2 days (37 °C, 5% CO₂, 200 rpm).
11. Measure the optical density (OD) using a spectrophotometer to monitor bacterial growth curves until the absorbance at OD₆₂₀ reaches to 2.0.
12. Separate the bacterial pellets and the conditioned media by centrifuging at 1,000 x g for 15 min. Wash the collected bacterial pellets with 1x PBS and resuspend in 4 mL of RPMI 1640 supplemented with 10% fetal bovine serum. Do not include any antibiotics in the medium.
13. Maintain the bacterial pellets in RPMI and incubate in a shaker incubator for 4 h at 37 °C with 5% CO₂ at a speed of 100 rpm.
14. For the preparation of the probiotic supernatant, remove the bacterial pellet via centrifugation at 1000 x g, for 15 min at 4 °C. Sterile-filter the recovered supernatant using a 0.22 μm filter and store at −80 °C until use.

2. Generation of spheroids

1. Preparing colorectal cancer cell lines​
   1. Grow DLD-1, HT-29, and WiDr cell lines as monolayers until 70-80% confluency and incubate the plate at 37 °C in a 5% CO₂ incubator (Growth medium: RPMI containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin).
   2. For cells grown in 100 mm Petri dish, wash the plate twice with 4 mL of 1x PBS. Add 1 mL of 0.25% trypsin-EDTA and incubate the Petri dish for 2 min at 37 °C in a 5% CO₂ incubator to dissociate the cells.
   3. After incubation, check for the cell dissociation under a microscope and neutralize trypsin-EDTA with 5 mL of growth medium.
   4. Transfer the dissociated cells to a 15 mL conical tube and centrifuge for 3 min at 300 x g.
   5. Discard the supernatant and resuspend gently with 3 mL of growth media.
   6. Count the cells with trypan blue to determine viable cells using a hemocytometer. (Figure 1B (i))
2. Spheroid formation​
   1. In a 15 mL conical tube, dilute the cells from 2.1.5 to obtain 1 – 2 x 10⁵ cells/mL (Figure 1B (ii))
   2. Add final concentration of 0.6% methylcellulose to the cell suspension and transfer the diluted cells to a sterile reservoir.
      ​NOTE: For each cell line, the amount of methylcellulose needed should be titrated and determined accordingly.
   3. Use a multichannel pipette to dispense 200 µL of cells to each well of an ultra-low attachment 96-well round bottom microplate. (Figure 1B (iii))
   4. Incubate the plate at 37 °C in a 5% CO₂ incubator for 24 – 36 h.
   5. After 24 – 36 h, observe the plate under a light microscope to ensure spheroid formation.

3. Treating 3D colorectal cancer cells with LCFS

1. Generate spheroids as described in steps 2 and 3.
2. Before performing the LCFS treatment, thaw the frozen LCFS at room temperature (RT) for 10 – 20 min.
3. Inoculate the LCFS stock solution into a growth medium. Serially dilute to 25%, 12.5%, and 6% in the growth medium (i.e., 25% LCFS = 150 µL of growth medium + 50 µL of LCFS).
4. Take out the cell culture plate containing spheroids from the incubator and remove as much of the growth medium as possible from each well using a 200 μL pipette.
5. Add the growth media with LCFS on the cells and incubate at 37 °C in a 5% CO₂ incubator for 24 – 48 h.
   NOTE: The volume to be used will depend on the plate size as follows: 2 mL for 6-well cell culture plates; 200 μL for 96-well cell culture plates.

Representative Results

Figure 1: Schematic representation of spheroid formation and LCFS preparation.

(A) Schematic representation images of the LCFS generation protocol are marked by (i-iv) (B) Schematics of the methylcellulose-mediated spheroid formation are marked by (i-iii).

Materials

Applied Biosystems MicroAmp Optical Adhesive Film	Thermo Fisher Scientific	4311971	100 covers
Axygen 2.0 mL MaxyClear Snaplock Microcentrifuge Tube, Polypropylene, Clear, Nonsterile, 500 Tubes/Pack, 10 Packs/Case	Corning	SCT-200-C	500 Tubes/Pack, 10 Packs/Case
BD Difco Bacto Agar	BD	214010	500 g
BD Difco Lactobacilli MRS Broth	BD	DF0881-17-5	500 g
Corning Phosphate-Buffered Saline, 1X without calcium and magnesium, PH 7.4 ± 0.1	Corning	21-040-CV	500 mL
Falcon 5 mL Round Bottom Polystyrene Test Tube, with Cell Strainer Snap Cap	Corning	352235	25/Pack, 500/Case
Fetal Bovine Serum, certified, US origin	Thermo Fisher Scientific	16000044	500 mL
Lactobacillus fermentum	Korean Collection for Type Cultures	KCTC 3112
L-Cysteine hydrochloride monohydrate	Sigma-Aldrich	C6852-25G	25 g
Methyl Cellulose (3500-5600mPa·s, 2% in Water at 20°C)	TCI	M0185	500 g
MicroAmp Fast Optical 96-Well Reaction Plate with Barcode, 0.1 mL	Applied Biosystems	4346906	20 plates
Millex-GS Syringe Filter Unit, 0.22 µm, mixed cellulose esters, 33 mm, ethylene oxide sterilized	Millipore	SLGS033SB	250
Penicillin-Streptomycin (10,000 U/mL)	Thermo Fisher Scientific	15140122	100 mL
RPMI-1640	Gibco	11875-119	500 mL
Trypsin-EDTA (0.25%), phenol red	Thermo Fisher Scientific	25200056	100 mL
Materials/Equipment/Software
CO2 incubator	Thermo fisher	HERAcell 150i
Conical tube 15 ml	SPL	50015
Conical tube 50 ml	SPL	50050
Corning Costar Ultra-Low Attachment Multiple Well Plate	Sigma-Aldrich	CLS7007
Corning Costar Ultra-Low Attachment Multiple Well Plate	Sigma-Aldrich	CLS3471
Costar 50 mL Reagent Reservoirs, 5/Bag, Sterile	Costar	4870
Countess Cell Counting Chamber Slides	Thermofisher	C10228
Countess II FL Automated Cell Counter	invitrogen	AMQAF1000
EnSpire Multimode Reader	Perkin Elmer		Enspire 2300
Eppendorf Research Plus Multi Channel Pipette, 8-channel	Eppendorf	3122000051
Incubated shaker	Lab companion	SIF-6000R
Multi Gauge Ver. 3.0,	Fujifilm		Tokyo, Japan
Optical density (OD)LAMBDA UV/Vis Spectrophotometers	Perkin Elmer		Waltham, MA, USA
Phase-contrast microscope	Olympus		Tokyo, Japan
SPL microcentrifuge tube 1.5mL	SPL	60015
SPL Multi Channel Reservoirs, 12-Chs, PS, Sterile	SPL	21012
Vibra-Cell Ultrasonic Liquid Processors	SONICS-vibra cell	VC 505	500 Watt ultrasonic processor
Vinyl Anaerobic Chamber	COY LAB PRODUCTS