Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Aug:129 Suppl 142:1-30.
doi: 10.1111/apm.13168.

Characterization of in vitro 3D cultures

Nabi Mousavi  1
Affiliations

Characterization of in vitro 3D cultures

Nabi Mousavi. APMIS. 2021 Aug.

Abstract
in English, Danish

Over the past decade, 3D culture models of human and animal cells have found their way into tissue differentiation, drug development, personalized medicine and tumour behaviour studies. Embryoid bodies (EBs) are in vitro 3D cultures established from murine pluripotential stem cells, whereas tumoroids are patient-derived in vitro 3D cultures. This thesis aims to describe a new implication of an embryoid body model and to characterize the patient-specific microenvironment of the parental tumour in relation to tumoroid growth rate. In this thesis, we described a high-throughput monitoring method, where EBs are used as a dynamic angiogenesis model. In this model, digital image analysis (DIA) is implemented on immunohistochemistry (IHC) stained sections of the cultures over time. Furthermore, we have investigated the correlation between the genetic profile and inflammatory microenvironment of parental tumours on the in vitro growth rate of tumoroids. The EBs were cultured in spinner flasks. The samples were collected at days 4, 6, 9, 14, 18 and 21, dehydrated and embedded in paraffin. The histological sections were IHC stained for the endothelial marker CD31 and digitally scanned. The virtual whole-image slides were digitally analysed by Visiopharm® software. Histological evaluation showed vascular-like structures over time. The quantitative DIA was plausible to monitor significant increase in the total area of the EBs and an increase in endothelial differentiation. The tumoroids were established from 32 colorectal adenocarcinomas. The in vitro growth rate of the tumoroids was followed by automated microscopy over an 11-day period. The parental tumours were analysed by next-generation sequencing for KRAS, TP53, PIK3CA, SMAD4, MAP2K1, BRAF, FGFR3 and FBXW7 status. The tumoroids established from KRAS-mutated parental tumours showed a significantly higher growth rate compared to their wild-type counterparts. The density of CD3+ T lymphocytes and CD68+ macrophages was calculated in the centre of the tumours and at the invasive margin of the tumours. The high density of CD3+ cells and the low density of CD68+ cells showed a significant correlation with a higher growth rate of the tumoroids. In conclusion, a novel approach for histological monitoring of endothelial differentiation is presented in the stem cell-derived EBs. Furthermore, the KRAS status and density of CD3+ T cells and macrophages in the parental tumour influence the growth rate of the tumoroids. Our results indicate that these parameters should be included when tumoroids are to be implemented in personalized medicine.

I løbet af det seneste årti er man begyndt at bruge 3D cellekulturer med humane og dyreceller til vaevsdifferentieringsstudier, til medicinudvikling, til personaliseret medicin og til cancer mikromiljøstudier. Embryoid bodies (EBer) er in vitro 3D cellekulturer etableret fra muse stamceller, hvorimod tumoroider er 3D kulturer etableret fra patienters tumorceller. Formålet med denne PhD var at kortlaegge en ny anvendelse af ”embryoid body” modellen samt at karakterisere sammenhaengen mellem det patient-specifikke tumormiljø og tumoroidernes efterfølgende vaekst in vitro. I denne PhD har vi kortlagt en high throughput monitoreringsmetode, hvor EBerne er anvendt som en dynamisk angiogenesemodel, og hvor immunfarvede snit af EBerne er analyseret digitalt. Herudover har vi undersøgt sammenhaengen mellem den genetiske profil og det inflammatoriske mikromiljø i primaertumorerne med vaekstraten af de fremdyrkede tumoroider. EBerne blev dyrket i spinnerflasker og høstet på dag 4, 6, 9, 14, 18, og 21, dehydreret og indstøbt i paraffin. De histologiske snit blev farvet for en endothel markør (CD31) og skannet digitalt. Disse hel-slide billeder blev analyseret ved hjaelp af Visiopharm® software. Den histologiske evaluering viste, at der blev dannet vaskulaer-lignende strukturer med tiden. Den kvantitative digitale billedanalyse fandt vi velegnet til at monitorere et vaekstareal samt endotheldifferentieringen i EBerne. Tumoroider blev etableret fra 32 colorektal adenokarcinomer. In vitro vaeksten af tumoroiderne blev fulgt i 11 dage ved hjaelp af et automatiseret mikroskop. De oprindelige tumorer blev analyseret ved hjaelp af next generation sekventering for KRAS, TP53, PIK3CA, SMAD4, MAP2K1, BRAF, FGFR3 og FBXW7 mutationer. Tumoroider, som blev etableret fra primaertumorer indeholdende en KRAS mutation, viste en signifikant højere vaekstrate sammenlignet med wild-type tumorer. CD3+ T celler og CD68+ makrofagers densitet blev beregnet i tumormidten samt i den invasive margin af tumorerne. Vi fandt en statistisk signifikant sammenhaeng mellem vaeksten af tumoroiderne in vitro og taetheden af CD3+ immunceller og lav taethed af CD68+ immunceller i primaertumoren. Vi konkluder, at en kombination af immunfarvning, digital slideskanning og digital billedeanalyse er en ny tilgang til at følge endothel uddifferentiering i en EB model. Derudover påvirker KRAS mutationsstatus samt antallet af CD3+ T celler og CD68+ makrofager i primaervaevet tumoroidernes efterfølgende vaekst. Vores resultater indikerer, at disse parametre bør analyseres, når tumoroider ønskes anvendt til personaliseret medicin.

Keywords: In vitro 3D culture; digital pathology; embryoid body; tumoroid.

PubMed Disclaimer

References

REFERENCES

    1. Ekert JE, Johnson K, Strake B, Pardinas J, Jarantow S, Perkinson R, et al. Three-dimensional lung tumor microenvironment modulates therapeutic compound responsiveness in vitro-implication for drug development. PLoS One. 2014;9(3):e92248.
    1. Luca AC, Mersch S, Deenen R, Schmidt S, Messner I, Schäfer K-L, et al. Impact of the 3D microenvironment on phenotype, gene expression, and EGFR inhibition of colorectal cancer cell lines. PLoS One. 2013;8(3):e59689.
    1. Pampaloni F, Reynaud EG, Stelzer EH. The third dimension bridges the gap between cell culture and live tissue. Nat Rev Mol Cell Biol. 2007;8(10):839-45.
    1. Birgersdotter A, Sandberg R, Ernberg I. Gene expression perturbation in vitro-a growing case for three-dimensional (3D) culture systems. Semin Cancer Biol. 2005;15(5):405-12.
    1. Ravi M, Paramesh V, Kaviya SR, Anuradha E, Solomon FD. 3D cell culture systems: advantages and applications. J Cell Physiol. 2015;230(1):16-26.