Name: Hepatocellular Carcinoma Organoid Culture and Digestion to Study Liver Cancer Pathogenesis
Uploaded: 2023-08-18T14:50:00
Description: Watch this Scientific Journal Video about Hepatocellular Carcinoma Organoid Culture and Digestion to Study Liver Cancer Pathogenesis at JoVE.com

hepatocellular carcinoma organoid culture and digestion to study liver cancer pathogenesis

Optimering af HCC-organoiddannelse til målidentifikation og lægemiddeludvikling

Hepatocellular carcinoma (HCC), a prevalent and extensively diverse tumor1, has garnered considerable attention within the medical community. The presence of lineage plasticity and substantial heterogeneity in HCC suggests that tumor cells originating from various patients and even distinct lesions within the same patient may manifest dissimilar molecular and phenotypic traits, thereby presenting formidable obstacles in the advancement of innovative therapeutic approaches2,3,4,5. Consequently, there is an imperative need for enhanced comprehension of the biological attributes and mechanisms of drug resistance in HCC to inform the formulation of more efficacious treatment strategies.
In recent decades, researchers have dedicated their efforts to the development of in vitro models for the purpose of studying HCC3,4. Despite some advancements, limitations persist. These models encompass a range of techniques, such as the utilization of cell lines, primary cells, and patient-derived xenografts (PDX). Cell lines serve as in vitro models for long-term culture of tumor cells obtained from HCC patients, offering the benefits of convenience and facile expansion. Primary cell models involve direct isolation and culture of primary tumor cells from patient tumor tissues, thereby providing a representation of biological characteristics that closely resemble those of the patients themselves. PDX models entail the transplantation of patient tumor tissues into mice, with the aim of more faithfully simulating tumor growth and response. These models have been instrumental in HCC research, yet they possess certain limitations, including the heterogeneity of cell lines and the inability to fully replicate in vivo conditions. Furthermore, prolonged in vitro cultivation may result in the deterioration of the cells&#39; original characteristics and functionalities, posing challenges in accurately representing the biological properties of HCC. Additionally, the utilization of PDX models is both time-consuming and costly3.
To address these limitations and more accurately replicate the physiological attributes of HCC, the utilization of organoid technology has been introduced as a promising research platform capable of surpassing previous constraints. Organoids, which are three-dimensional cell models cultured in vitro, have the ability to replicate the structure and functionality of actual organs. However, in the context of HCC, there exist certain challenges in establishing organoid models. These challenges include insufficiently detailed descriptions of HCC organoid construction procedures, a lack of comprehensive protocols for the entire process of HCC organoid construction, and the typically small size of cultured organoids6,7,8. In light of the typically limited dimensions of cultured organoids, we endeavored to tackle these challenges through the development of a comprehensive protocol encompassing the entirety of HCC organoid construction6. This protocol encompasses tissue dissociation, organoid plating, culture, passaging, cryopreservation, and resuscitation. By optimizing the procedural steps and refining the composition of the culture medium, we have successfully established HCC organoid models capable of sustained growth and long-term passaging6,8. In the subsequent sections, a comprehensive account of the operational intricacies and pertinent factors involved in the construction of HCC organoids will be presented.

Forfatter Spotlight: Undersøgelse af leverkræftpatogenese ved hjælp af patientafledte organoider 

Udvikling og optimering af en human hepatocellulært carcinom patientafledt organoidmodel til potentiel målidentifikation og lægemiddelopdagelse

Our lab aims to investigate the molecular pathogenesis of liver cancer and identify novel therapeutic targets for drug development. We would like to answer how cancer initiates, metastasis, and progress to drug tolerance utilizing novel disease models such as patient derived organoids. The current experimental challenges are in two aspects.First, there is a lack of complete set of protocols for constructing hepatocellular carcinoma organoids for the whole process. Second, the size of cultured HCC organoids is too small for subsequent experimental processing. In this protocol, we provide the details of the optimized organoids culture and subsequent transfer and freezing procedures, as well as the precautions to be taken during all the procedures.We optimized the composition of the organoid medium for organoids with close to the original tissue characteristics. We may monitor the tumor evolution using the patient-derived organoids and try to identify novel molecular and therapeutic targets for HCC treatment.

Watch this Scientific Journal Video about Hepatocellular Carcinoma Organoid Culture and Digestion to Study Liver Cancer Pathogenesis at JoVE.com

Hepatocellular Carcinoma Organoid Culture and Digestion to Study Liver Cancer Pathogenesis  

Hepatocellulært carcinom organoid kultur og fordøjelse for at studere leverkræft patogenese

For at udføre organoidbelægning fjernes supernatanten først fra centrifugen og vaskes hepatocellulære carcinomceller. Re-suspendere cellerne i 50 mikroliter kældermembranekstrakt eller BME pr. Brønd til plettering. Derefter suspenderes cellerne i avanceret DMEM / F12.Derefter pipetteres celleaggregaterne forsigtigt, indtil der ses en jævn suspension. Tilføj BME til suspensionen, og sørg for, at BME-koncentrationen er mellem 30 og 50%Nu frø 50 mikroliter BME-dråber med celleklynger i midten af en 24 brøndplade. Lad dråberne størkne ved 37 grader Celsius i 20 minutter.Tilsæt 500 mikroliter forvarmet medium til hver brønd og inkuber i en celleinkubator ved 37 grader Celsius. Opdater kulturmediet hver anden til tredje dag. Efter to uger udskiftes isolationsmediet med organoidekspansionsmediet.Efter syv til 10 dages dyrkning, når organoiderne har nået den passende tæthed, behandles kulturerne efter behov. For at passere organoiderne skal du først forvarme ultra lav fastgørelsesoverfladecellekulturplader i en inkubator. Optø derefter den frosne BME natten over ved fire grader Celsius indtil lige før brug.Forvarm organoidhøstopløsningen og trypsinerstatningen ved 37 grader Celsius i 30 minutter. Efter forberedelsesfasen fjernes kulturmediet fra den organoide kulturplade. Overfør derefter organoidsuspensionen til et 15 ml rør.Tilføj nu organoid høstopløsning i forhold til mængden af BME. For at blande suspensionen skal du bruge en 1000 mikroliter pipettepistol til at skrabe og pipette op og ned. Efter inkubation af røret ved stuetemperatur i 30 minutter skal du bruge en milliliter pipette til forsigtigt at aspirere BME.Sørg for, at BME er helt opløst. Overvåg ekstraktet hvert 10. minut, indtil en klar organoid celleklynge er synlig. Derefter centrifugeres ved stuetemperatur ved 400 g i fem minutter.Så meget af supernatanten fjernes som muligt. For at starte enzymatisk fordøjelse af hepatocellulære carcinomorganoider tilsættes en til fem ml af den forvarmede trypsinerstatning til den dyrkede organoidpellet. Inkuber suspensionen ved 37 grader Celsius i to minutter.Brug et mikroskop til at kontrollere, om organoider dissocieres i små klynger på to til 10 celler. For at stoppe fordøjelsen tilsættes et passende volumen koldt basalmedium. Derefter centrifugeres organoiderne ved 400 g i fem minutter ved otte grader Celsius.Fjern forsigtigt så meget supernatant som muligt. Re-suspender det ønskede antal organoider i den korrekte matrix til plettering. Hver 10. dag passerer organoiderne afhængigt af tætheden af organoidvækst.HCC organoider sfæroider blev observeret inden for tre dage efter dyrkning. Kompakte sfæroider med afrundede kanter og permeabel cytosol blev set på den første etableringsdag. Organoider var af samme størrelse og havde den største diameter, når de blev dyrket i 30 til 50% BME.BME var mest fragmenteret med 10%hvilket resulterede i de mindste organoider. BME var mest intakt på 100%, men resulterede i organoider med mellemliggende diameter. Den prolifererende HCC-organoid opnåede en størrelse på over 500 mikrometer i hver kultur efter tre generationer.HCC-organoider af væsentlig størrelse på mere end 1000 mikrometer blev opnået inden for en 20-dages dyrkningsperiode.

Research

JoVE Journal

Cancer Research

Hepatocellular Carcinoma Organoid Culture and Digestion to Study Liver Cancer Pathogenesis

Development and Optimization of A Human Hepatocellular Carcinoma Patient-Derived Organoid Model for Potential Target Identification and Drug Discovery

Hepatocellular carcinoma (HCC) is a highly prevalent and lethal tumor worldwide and its late discovery and lack of effective specific therapeutic agents ...