En studie publicerad i onsdags ,12/4, visar att vi rör oss längre och längre ifrån att behöva använda djur i forskningssyfte. Att behöva testa kemiska substanser på djur eller ens behöva ha djur i labb för syfte att ha tillgång till deras blod,celler,vävnader mm...vid forskning.En utveckling som djurrättsorganisationer har drivit i många år. Här i den nu aktuella studien utförd av 11 forskare och med stöd av studenter från ett flertal fakulteter visar man att studier vanligtvis utförda med material från djur helt har undvikits.
För att kunna bedriva mänsklig cellforskning behövs tillförda substanser som får cellerna att agera som inne i en levande kropp. En substans (derivat) kan vi slarvigt kalla näring,att mata cellerna med käk så de uppför sig så naturligt som möjligt. Dvs dela sig,fästa vid varandra,forma sig osv...
Olika substrat ingår i nåt man kallar medium.En blandning av olika derivat som möjliggör att cellerna både rör på sig och överlever förhoppningsvis så länge som behövs.
I majoriteten (alla?) av alla medium ingår en komponent tagen från djur, ett derivat kallat Foetal Bovine Serum (FBS). Derivatet är utvunnet från djurets blod och har ansetts högst tillämpbart som supplement vid cellforskning. Det för att det innehåller låga mängder av antikroppar (antibodies) och höga värden näring.
I majoriteten (alla?) av alla medium ingår en komponent tagen från djur, ett derivat kallat Foetal Bovine Serum (FBS). Derivatet är utvunnet från djurets blod och har ansetts högst tillämpbart som supplement vid cellforskning. Det för att det innehåller låga mängder av antikroppar (antibodies) och höga värden näring.
Som jag förstår det är FBS taget ur blodet från ett levande foster hos ett dräktigt djur man precis avlivat.
Mer specifikt tas det ur fostrets fortfarande pumpande hjärta.Och det tas utan att bedöva fostret. Etik?
Användande av FBS har dock på senare tid blivit kontroversiellt. Först ur ovanstående etiska perspektiv (djur - forskning-grymhet),men även huruvida FBS kan påverka forskningsresultatet i negativ aspekt.
Utan att gå för djupt in i detta område hoppar jag till nu aktuell studie. Forskarna har utvecklat ett medium utan tillsatser av FBS, vilket är tämligen spektakulärt. Först för att vi tar ytterligare ett steg från djurförsök vilket djurrättsorganisationer kämpat för i så många år, men även kommer ha betydelse för cellforskningen som helhet. Studiens innehåll berättar att detta medium kan användas för att bygga upp cellbanker. Att exempelvis celler från bröstcancer kan fås att "leva" och därmed kunna användas vid upprepade studier.
Jag går inte in på vad detta medium innehåller då det är alltför komplicerat för undertecknad. Men kan konstatera att forskarna flyttat gränserna för cellforskningen högst anmärkningsvärt. Var kommer då HoloMonitor in i bilden kanske ni undrar? Jo,först för att forskarna varit flitiga att använda tekniken genom hela studieförloppet. Man har använt sig av 2 st HoloMonitor. M3 och M4. Här nedan redogör jag för deras användande. Sen tanken med att bygga upp cellbanker med levande celler kopplar jag till forskares växande intresse att studera celler utan infärgning. Att hålla celler vid liv så långt möjligt via dessa cellbanker. För att helt kunna undvika infärgning men ändå ha möjlighet att studera cellerna är tekniken HoloMonitor erbjuder ypperlig. Inte minst bevisar forskarna det i denna studie. Hänger ni med i resonemanget?
Men till studien,som är betitlad :
(Fetningar och understryk är mina egna)
Abstract
Cell culturing methods are increasingly used to reduce and replace the use of living animals in biomedical research and chemical toxicity testing. Although living animals are avoided when using cell culturing methods, they use animal-derived components of which one of the most commonly used is foetal bovine serum (FBS).
FBS is added to cell culture media among other supplements to support cell attachment/spreading and cell proliferation. The safety, batch-to-batch variation, and ethical problems with FBS are acknowledged and therefore world-wide efforts are ongoing to produce FBS free media.
Here, we present the composition of a new defined medium with only human proteins either recombinant or derived from human tissues.
This defined medium supports long-term culturing/routine culturing of normal cells and of cancer cells, and can be used for freezing and thawing of cells, i.e. for cell banking.
Here, we show for our defined medium, growth curves and dose response curves of cells grown in two and three dimensions, and applications such as cell migration.
Cell morphology was studied in real time by phase contrast and phase holographic microscopy time-lapse imaging.
The cell lines used are human cancer-associated fibroblasts, keratinocytes, breast cancer JIMT-1 and MDA-MB-231 cells, colon cancer CaCo-2 cells, and pancreatic cancer MiaPaCa-2 cells as well as the mouse L929 cell line.
In conclusion, we present the composition of a defined medium without animal-derived products which can be used for routine culturing and in experimental settings for normal cells and for cancer cells, i.e. our defined medium provides a leap towards a universal animal product free cell culture medium.
2. Materials and Methods (urval)
2.9. Time-lapse videos of general cell movement
For time-lapse imaging, two different microscopes were used: a HoloMonitor® M3 phase holographic microscope (M3) (PHI AB, Lund, Sweden), with a 10 times phase contrast objective, placed in an incubator at 37 °C with air as atmosphere and a HoloMonitor® M4 phase holographic microscope (M4) (PHI AB) placed in a regular 37 °C CO2 incubator with 5% CO2 in air. For time-lapse imaging of routine cultures in the M3 microscope, the cells were seeded in a 25 cm2 Primaria® tissue culture flask, which was incubated for 24 hours in a 37 °C CO2 incubator.
When removed from the incubator, the lid of the flask was closed tightly to preserve the 5% CO2 in air atmosphere and the flask was placed on the stage of the M3 microscope. Images were captured every 10 minutes for 72 hours. For time-lapse imaging of cells in the M4 microscope, cells were seeded in 6-well Primaria® plates. The cells were allowed to attach for 24 hours prior to initiation of the time-lapse imaging. The standard lid of the 6-well plate was replaced with three HoloLid® 71120 for 6-well plates (PHI AB). The 6-well plate was then placed on the motorised stage of the M4. For imaging in the M3 and M4 microscopes, the software programs Hstudio® (PHI AB) and HoloMonitor® Appsuite (PHI AB) were used, respectively. In the M4 microscope, images were acquired at 6-8 locations per well, at 5 minute intervals for 72 hours. Only one location was imaged in the M3 microscope.
2.10. Cell movement from spheroids
Movement of cells from spheroids was analysed using the M3 and M4 microscopes. Spheroids seeded in Primaria® 6-well plates were analysed using the M4 microscope while spheroids seeded on linear fibres of PCL were analysed in the M3 microscope.
For analysis in the M4 microscope, the spheroids were added to 6-well Primaria® plates. Before adding the spheroids, a small area (0.5 cm2) in the centre of each well was incubated with a small drop of medium for 1 hour. The spheroids were added to the drop with a micropipette. To prevent drying out, 1 ml of medium was added along the wall of each well. Capillary action prevented the medium from flowing to the drop. After 24 hours of incubation, medium was added to a final volume of 4 ml. The standard lid of the Petri dish was replaced with three HoloLid® 71120 for 6-well plates. Spheroids were also subjected to treatment with 100 nM paclitaxel. The 6-well plate was then placed on the motorized stage of the M4 microscope. The edge of the spheroid to be imaged was kept in focus at the edge of the viewing field using the application Kinetic Motility Assay in HoloMonitor® App Suite and imaging every 5 min for 72 hours was initiated. The Wound Healing application in HoloMonitor® App Suite was used to analyse cell movement from the spheroids.
For analysis in the M3 microscope, spheroids were seeded on linear fibres of biocompatible PCL [42]. Linear fibres of PCL were obtained as described in Malakpour-Permlid et al. [42] with the difference that a rotating collector was used, and the time of electrospinning was carefully controlled to obtain a single layer of parallel fibres. The fibres were spun on a plastic backing and this was cut into 1 cm2 pieces.
Then, the lid was tightened, and the flask moved to the stage of the M3 microscope. Images were captured every 10 minutes for 72 hours.
3.4. Time-lapse imaging
In general, cells are only viewed statically in a phase contrast microscope and therefore information about cell behaviour in between microscopic evaluation is less known. We have studied cell behaviour during several days by time-lapse imaging in M3 or M4 holographic microscopes (Table 5 lists the videos shown in Supplemental information).
Supplemental Video S1 shows an M3-derived phase contrast microscope time-lapse video of keratinocytes dancing in defined medium (real time 72 hours). Single keratinocytes and keratinocytes in groups show circular movement where they appear to search for each other and make larger groups of cells. We also have videos from the M4 microscope showing the same behaviour (data not shown). The Supplemental Video S2 shows a phase holographic time-lapse video of CaCo-2 cells in defined medium (real time 72 hours). The CaCo-2 cells do not move around substantially. Several cell divisions are seen. The CaCo-2 cells are not optimal for phase holographic imaging as they are very thin, and the entire cells are actually not seen. Phase contrast microscopy shows that the cells form a tight sheet and each cell has a rather large cytoplasm (supplemental Figure S2, videos from the M3 microscope show the same behaviour (data not shown)). Holographic microscopy shows the thicker parts of cells i.e. the cell nuclei and cytoplasm close to the nuclei, but not the very thin parts of the cytoplasm. This is a well-known phenomenon related to phase holographic imaging, since the phase shift of the light in very thin parts of a cell is too small to create an interference pattern or hologram (www.phiab.com). Supplemental Video S3 (phase holographic time-lapse) shows well-defined elongated MDA-MB-231 cells moving around at high speed, and several divisions where the cells round up are seen. Supplemental Video S4 (phase holographic time-lapse) shows well-defined JIMT-1 cells. All these videos show the behaviour of cells in routine passage.
We have used the M4 to study the movement of cells from spheroids seeded on a flat surface. We were interested in investigating if cell movement was affected by the presence of CAFs which has been demonstrated by others using medium supplemented with 10% FBS [16], [47], [61] and we have also treated spheroids with paclitaxel. The behaviour of cells in JIMT-1 spheroids (Supplemental Video S5), in MiaPaCa-2 spheroids (Supplemental Video S6), in JIMT-1/CAF spheroids (Supplemental Video S7), and in MiaPaCa-2/CAF spheroids (Supplemental Video S8) was investigated with the M4 microscope.
In our study, spheroids were formed by seeding cancer cells alone or together with CAFs in the defined medium in hydrophobic Petri dishes and incubating them on a rotary shaker placed in a 37 °C CO2 incubator. CAFs have been shown to have a supporting role in the tumour microenvironment stimulating cancer cell proliferation, invasion, and metastasis, imparting resistance to cancer therapeutic drugs as well as stimulating angiogenesis [35]. By time-lapse imaging using the M4, we monitored the rate of cell migration from the spheroid edges. Others have shown that CAFs affect the rate of cancer cell migration in model systems using medium supplemented with 10% FBS [16], [47], [61]. Here we show that CAFs increased the rate of cell migration from spheroids containing CAFs and cancer cells compared to the rate of migration from spheroids with only cancer cells using the defined medium. Furthermore, we show that CAFs modulate the toxicity of paclitaxel. Here, we show proof-of-principle that it is possible to form spheroids with cancer cells only or together with CAFs in the defined medium and to use the spheroids in different assays and applications. /
I forskningsrapporten har man bifogat ett helt gäng med HoloMonitorfilmer ni kan titta på.
I sammanfattningen i slutet av studien skriver man :
Although the scientific and ethical problems with FBS are widely recognised, FBS is still extensively used in cell culture media. Defined medium has been developed and is used in various cellular models (some examples: [29], [60], [4]; see FCS-free database fcs-free.org) but routine passaging is performed in medium supplemented with FBS.
Our approach was to develop a universal defined medium with human proteins that could be used for reproducible routine passaging and for cell banking as well as in different experimental settings.
The relevance of the defined medium is proven with several cell types (normal and cancer) cultured in 2D and 3D, using end points such as growth curves, dose response curves, cell migration, and effects of known reference chemicals.
Here, we present the composition of the defined medium which instead of serum contains human proteins for cell attachment/spreading and stimulation of cell proliferation. We used this medium in five practical sessions with L929 cells and keratinocytes in the course BIOR21 Toxicology at the Department of Biology, Lund University in the spring of 2022.
Twenty-four students performed growth curve experiments, dose response experiments, wound healing experiments, live/dead cell assays, and they also successfully used the keratinocytes in the OECD skin sensitisation assay TG442D (data not shown).
In conclusion, here we present the composition of a medium free of FBS with a defined content of chemicals and human proteins. The defined medium showed to be adequate for routine culturing, including freezing and thawing for a number of different normal and cancer cell lines. In addition, we have shown that the defined medium can be used in different experimental settings such as to obtain growth curves, dose response curves in 2D and 3D, and to study cell migration in different contexts. The validity of the defined medium was confirmed by comparing with data obtained by culturing cells in FBS- or DHHS-supplemented medium. We hope that the piece of work presented here will contribute to further development of universal media without animal components.
Acknowledgements
Credit to all students of BIOR21 Toxicology, Department of Biology, Lund University, the Spring of 2022 who worked with 12 different compounds investigating toxicity in five different practical trainings using L929 cells and keratinocytes with the defined medium. Their growth curve data for the L929 cells is included in this article. Credit to students in BIOR21 (Toxicology) and BIMM18 (Cell Culturing) courses at the Department of Biology, Lund University, Sweden during 2015-2021 that have contributed to non-published data.
This research could not have been done without funding from Forska Utan Djurförsök (forskautandjurforsok.se), and donations through crowd funding by Carolina le Prince and the Kalenderflickorna, Bröstcancerföreningen Pärlan Helsingborg (helsingborg.brostcancerforbundet.se), Bröstcancerföreningen Viktoria Ängelholm (angelholm.brostcancerforbundet.se), and Bröstcancerföreningen Victoria Östra Skåne (ostraskane.brostcancerforbundet.se), Stig and Lisa Ekelund, and Mari-Ann and Brainerd Lindberg via Lund University Development Office. The sponsors have no other role than providing funding. We wish to thank Prof. Tuula Heinonen (Tampere University, Finland) for constructive remarks regarding the manuscript and Kerstin Petti for English editing.
Min kommentar
Denna studie som är ledd av HoloMonitorfrälste professor Stina Oredsson är imponerande både sett till uppdragets mål att försöka hitta ett alternativ till FBS som till att de faktiskt lyckades med uppsåtet. Sen att alla forskare samt studenter enträget använt sig av 2 versioner av HoloMonitorinstrumentet genom hela studien för att komma vidare till nästa steg,till nästa steg osv...är bara det ett bevis för teknikens användbarhet.Att de dessutom förespråkar cellstudier via erhållna cellbanker är en bonus sett till att forskare då kan minimera infärgning vilket leder fram till behovet av användning av ex HoloMonitor.
Jag gillar denna studie starkt (sett till procedur att använda levande fosterhjärtan) vilket förmodligen världens alla djurrättsorganisationer håller med mig om. Bra jobbat Stina et al. !
Mvh the99
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