A foundational approach to culture and analyze malnourished organoids

Figures & data

Figure 1. Malnourished media composition and study design. To generate malnourished organoids, certain media additives were eliminated to reduce nutritional content while maintaining cellular viability. Following establishment of media formulations to reduce protein, amino acid, and carbohydrate concentrations, both organoids and organoid-derived monolayers were incubated in the various media compositions and subsequently analyzed for the effects of malnourishment.

a. Malnourished media. The 50% L-WRN was essential to cellular viability and could not be removed or diluted. However, certain components of the ISC media could be removed without detrimental effects to cellular viability, and the volume was replaced with 1X PBS to generate 50% PBS-SC media. The standard media formulation (100%) is composed to 50% ISC media and 50% L-WRN media. The 75% malnourished media is composed of 25% PBS-SC, 25% ISC, and 50% L-WRN. The 50% media formulation is composed of 50% PBS-SC and 50% L-WRN. Overall, by diluting the ISC media, the concentration of protein, amino acids, and carbohydrates was reduced. All media types (100%, 75%, and 50%) also contained the inhibitors Y-27632 and A 83-01 in the L-WRN portion of the media.

b. Malnourished organoid and organoid-derived monolayer analyses.

Top: Organoids from three separate donors were cultured in the three different media formulations for seven days. The organoids were then analyzed for changes in phenotypic appearance, gene expression via qRT-PCR, and protein expression via immunofluorescence analysis and lactate dehydrogenase release, intestinal alkaline phosphatase secretion, and interleukin-8 secretion assays.

Bottom: The media formulations were also used to culture malnourished organoid-derived monolayers (mHIODEM). Following organoid generation in 100% media for seven days, organoids from the three separate donors were trypsinized, the cells were seeded onto transwells, and the monolayers were cultured for 13 days in the three different media formulations. The monolayers were differentiated with DAPT for the last 2 days of culturing (starting on day 11). Afterwards, the monolayers were analyzed for permeability via TEER and FITC-dextran passage, as well as susceptibility to infection via S. flexneri invasion analyses.

Figure 2. Phenotypic analysis of malnourished organoids.

a. Phenotypic evaluation. Following culturing in the three media formulations, organoids derived from three separate donor samples (D11, D14, and D15) were evaluated phenotypically for changes in appearance. Overall and despite the reduced nutrient concentrations in the malnourished media, organoids matured and developed the characteristic sphere shape. Images above were examined at 5X (top) and 20X (bottom) magnifications for all three sets of samples. The images represent three independent experiments, each with two technical duplicates, for the three organoid lines. The data indicate that organoid development in malnourished conditions is consistent across the three separate donor samples.

b. Lactate dehydrogenase (LDH) release. Culture supernatants were collected from D11, D14, and D15 organoids following seven days of culture in the 100%, 75%, or 50% media formulations. The data represent the average of four biologically independent experiments from all three lines ± the standard errors of the mean (SEM), and are plotted relative to the positive lysis control provided by the kit. All samples had significantly less LDH release relative to the positive control (**, p <.01; ***, p <.001). There were no significant differences in LDH release across the organoid media formulations.

c. To examine cellular viability, D14 organoids were cultured in the three media formulations for seven days. Subsequently, the organoids were trypsinized and stained with trypan blue to obtain total cell numbers and the percent viability of the cells, calculated as the percent of live cells relative to the total number of cells. The 50% media formulation had reduced total cell counts (*, p <.05) compared to the 100% and 75% media formulations. However, there were no significant differences in the percent viability across the treatments. For both analyses, data represent the average of three biologically independent experiments ± the SEM, and each experiment had three technical replicates.

Figure 3. Gene expression analyses of malnourished organoids and patient biopsy samples with different BMIs. Quantitative RT-PCR was performed for the indicated genes. Data bars represent the average fold change ± the SEM. All data were normalized to the expression of the 18S housekeeping gene for each experiment. Statistical significance was determined with the Student’s T-test of the ΔCT and the treatment comparisons that are statistically different are indicated for each gene and comparison. Data were considered significant at a p value of < .05 (*, <.05; **, <.01; ***, <.001). Please note the different y-axes for each graph.

a. Malnourished organoids. Data are expressed as the average fold change relative to the 100% media formulation. Data for each gene represent expression values from the D11, D14, and D15 organoid lines, each cultured in the 100%, 75%, and 50% media formulations. A total of three biologically independent experiments were performed, and each experiment had technical duplicates.

b. Biopsy samples from low and high BMI individuals. Data are expressed as the average fold change relative to the healthy control biopsy samples. Gene expression data for both the healthy control and low BMI samples were collected from three independent donors (biological replicates), while the high BMI samples was from one donor. Each experiment had technical duplicates.

Figure 4. Protein expression in the malnourished organoids. Analyses were performed as a measure of protein expression in the organoids with the various media formulations.

a. Immunofluorescence analysis. D14 organoids were cultured for seven days in the 100%, 75%, or 50% media formulations and subsequently processed and stained for microscopy. The colors of each antibody or DNA stain (blue) are indicated. Muc2 (green) expression, which was analyzed with and without ZO-1 (red) staining to help differentiate secreted Muc2 versus Muc2 localized to goblet cells, was induced in the 75% and 50% media formulations. SpiB (green) expression was induced in the 50% media formulation. The immunofluorescence confirms the gene expression analyses. Please note the Muc2 and ZO-1 images are magnified so the red ZO-1 signal can be seen. All images were captured with a 60X objective.

b. Intestinal alkaline phosphatase (IAP). Culture supernatants were collected after seven days of culture from the D11, D14, and D15 organoid lines and measured in four biologically independent experiments. The average absorbance reading ± the SEM for each media formulation is plotted relative to the 100% control media. Statistical significance was determined by a Student’s T-test, and data were considered significant at a p value of < .05 (*, <.05; **, <.01; ***, <.001). There was a significant decrease between the 100% and 50% media formulations, as well as the 75% and 50% media. There was no significant difference between the 100% and 75% media.

c. Interleukin-8 (IL-8) secretion. Culture supernatants were analyzed from biological and technical replicates as noted above, with the same statistical analyses and significance considerations. There was a significant difference between the 100% and 50% media, as well as the 75% and 50% media formulation. There was no significant difference between the 100% and 75% media formulation.

Figure 5. Malnourished monolayer permeability analysis. To measure integrity and permeability of the malnourished monolayers, mHIODEM were derived from the D11 and D14 organoid lines, and TEER and diffusion of FITC-dextran were measured. Each measurement occurred following monolayer differentiation with DAPT. Data represent three biologically independent experiments, each with at least two technical replicate wells. For both analyses, the average data ± SEM are plotted. Statistical significance was determined with the Student’s T-test comparing results from the 100% media to the 75% or 50% media. Data were considered significant at a p value of < .05 (*, <.05; **, <.01; ***, <.001). Please note the different y-axes for each graph.

a. Transepithelial electrical resistance (TEER) was evaluated as a measure of resistance multiplied by the area of the transwell (Ω*cm²). Data are plotted relative to the 100% media condition, which is set at 100%. There was a significant decrease in TEER for the 75% (**, p < .01) and 50% (***, p < .001) media formulations, indicating higher transcellular and paracellular permeability under malnourished conditions. There was also a decrease between the 75% and 50% media, but the p value was .07.

b. FITC-dextran passage was evaluated as a measure of paracellular permeability by examining diffusion of the 4 kilodalton FITC-dextran molecule. There was a significant increase in FITC-dextran passage in both the 75% and 50% media formulations (*, p < .05), indicating higher paracellular permeability of the malnourished monolayers. There was also an increase in the 50% media relative to the 75% media, but the difference was not significant.

Figure 6. Malnourished monolayer infection analysis. Organoids were trypsinized, seeded onto transwells, and cultured in the 100%, 75%, and 50% media formulations. Following 11 days of culturing and a 48-hour differentiation treatment with DAPT, monolayers were subsequently infected with S. flexneri strain 2457T. Following apical administration of the bacteria, plates were incubated for 3 hours and then treated with gentamicin to kill the extracellular bacteria.

a. Phenotypic analysis of monolayers. Images were examined at 5X magnifications for pre-infection (top) and post-infection (bottom) monolayers for each media formulation. The images indicate that the monolayers can be established on transwells in the malnourished media conditions. Additionally, the malnourished media enabled a more consistent infection pattern across the monolayer in both the 75% and 50% media formulations, as opposed to a more site-specific infection patter in the 100% media formulation. Images are monolayers derived from the D14 organoid line and are representative of three biologically independent experiments with two technical replicates for each experiment.

b. Bacterial invasion rates were calculated by dividing the recovery titers (CFU/ml) by the infecting titers and multiplying by 100%. The average results are plotted relative to the 100% media formulation ± SEM. The data represent 4 biologically independent experiments (from organoid lines D14 (3 experiments) and D11 (1 experiment)), and each experiment had at least two replicate wells per condition. The Student’s T-test was used to measure significance, in which there was a significant increase in the invasion rate (*, p < .05) in monolayers cultured 75% media formulation relative to the 100% control media.

Table 1. Primers used in this study.

Gene	Forward Primer	Reverse Primer
18S	5’-AGAAACGGCTACCACATCCA-3’	5’-CCCTCCAATGGATCCTCGTT-3’
MUC2	5’-GCCAGCTCATCAAGGACAG-3’	5’-GCAGGCATCGTAGTAGTGCTG-3’
SPIB	5’-GGCCACACTTCAGCTGTCT-3’	5’- CCCCTGTAGCCATCCATTCC-3’
OCLN	5’-ACAAGCGGTTTTATCCAGAGTC-3’	5’-GTCATCCACAGGCGAAGTTAAT-3’
CLDN2	5’-ACCTGCTACCGCCACTCTGT-3’	5’-CTCCCTGGCCTGCATTATCTC-3’
MTOR	5’-ATGCTTGGAACCGGACCTG-3’	5’-TCTTGACTCATCTCTCGGAGTT-3’
ATG5	5’-AAAGATGTGCTTCGAGATGTGT-3’	5’-CACTTTGTCAGTTACCAACGTCA-3’
LYZ	5’-CTTGTCCTCCTTTCTGTTACGG-3’	5’-CCCCTGTAGCCATCCATTCC-3’
IAP	5’-CTTTGGTGGCTACACCTTGC-3’	5’-CTCGCTCTCATTCACGTCTGG-3’
LGR5	5’-CTCCCAGGTCTGGTGTGTTG-3’	5’-GAGGTCTAGGTAGGAGGTGAAG-3’

The 5’ to 3’ sequence of each primer pair (forward and reverse) are provided for each gene target.

Data availability statement

The authors confirm that the data supporting the findings of this study are available within the article.