Exploring the Effects of Adaptation to Serum-Free Cultivation for Adherent and Suspended HEK293 Cells

Abstract

Cellekultur er en samlebetegnelse på dyrking av celler utenfor organismen de opprinner fra. Dette er et felt med stor vitenskapelig og industriell relevans, blant annet for produksjon av biofarmasøytika og vaksiner, og for forskning på sykdomsmekanismer.

Tradisjonelt har dyrkingsmediet gjerne blitt supplementert med dyreserum for å fremme cellevekst, men dette har en rekke praktiske utfordringer inkludert variabel sammensetning og høy risiko for opprinner kontaminering. I tillegg har tilfeller av svindel i serumindustrien, samt større fokus på dyrevelferd, økt etterspørselen etter serumfrie alternativer.

Cellelinjer har blitt etablert fra en rekke ulike organismer, men bruk av humane celler er fordelaktig for produksjon tiltenkt menneskelig bruk. I denne studien ble den humane embryoniske nyrecellelinjen HEK293 tilpasset adherent vekst i det serumfrie mediet «FreeStyleTM 293 Expression Medium» ved hjelp av to ulike protokoller. Sammenligning av morfologi, vekst og levedyktighet antydet at de to tilpasningsmetodene hadde lignende effekt på cellene. Deretter ble en suspensjonskultur etablert fra hver av de ulikt tilpassede adherente cellegruppene. Tilsetning av antiklumpemiddel begrenset forekomsten av celleklynger, og sørget for at kulturene opprettholdt høy levedyktighet.

Analyse av intra- og ekstracellulære metabolitter, vekst, morfologi og levedyktighet separerte gruppene basert på vekstmodus og mediesammensetning, men ikke tilpasningmetode. Den største driveren av gruppeforskjellene viste seg overraskende nok å være at suspensjonscellene produserte itakonsyre – en metabolitt som vanligvis kun produseres av makrofager.

Cell culture – the cultivation of cells outside their organism of origin – is a field of high industrial and scientific relevance. Traditionally, cell culture media have been supplemented with serum to enhance growth. However, this has several practical drawbacks, including a high risk of contamination and composition variability. Cases of fraud in the serum industry, as well as the animal welfare aspect are additional factors driving the development of alternatives to serum-supplemented media.

Cell lines can be obtained from a variety of organisms, but for the production of biopharmaceuticals and vaccines cells of human origin are the preferred choice. HEK293 and its progeny are some of the most commonly used human cell lines today. The primary aim of this study was to adapt HEK293 cells to adherent growth in a serum-free medium using two different protocols, before comparing their effects on cell growth, morphology and metabolism. Next, the goal was to establish serum-free suspension cultures from the adapted cells and assess the impact of growth mode on the characteristics mentioned above.

Serum-free adherent HEK293 cell cultures were established in FreeStyleTM 293 Expression Medium through sequential and direct adaptation of HEK293 cells from complete growth medium. While the adapted cells demonstrated higher growth rates, lower adhesion capacity and a more rounded morphology than the cells in the serum-supplemented control, the manner of adaptation did not affect these parameters. Culture viability remained high for all groups throughout the process. Separate suspension cultures were initiated from the serum-free adherent cell groups and clustering was limited through supplementation with anti-clumping agent. No differences in growth dynamics or morphology were observed between the two suspension cultures, but their growth rates were lower than those of the serum-free adherent cells and the control.

Passage fluctuations in extracellular levels of glucose, lactate and ammonia were tracked for all cell groups. The first two metabolites were analyzed with 1H-NMR, while an ammonia kit was used for the latter. Production and consumption patterns of the three metabolites differed based on growth mode and medium composition, but not adaptation method. Endometabolome analysis of all cell groups was conducted with LC-MS/MS and capIC-MS/MS, identifying serum-free adherent cells, the control and the suspension cultures as three metabolically distinct. Remarkably high concentrations of itaconic acid were found in the suspension cells compared to the other groups.