Chemically diverse polyacrylate and polyacrylamide surfaces for human cardiomyocyte culture and their effect on phenotype
Human pluripotent stem cell (hPSC) derived cardiomyocytes can provide robust in vitro models for pharmaceutical drug screening and modelling cardiac disease. To fully realise these potentials, hPSC-cardiomyocytes must be reproducibly cultured to a more mature state than has thus far been achieved. D...
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| Format: | Thesis (University of Nottingham only) |
| Language: | English |
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2014
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| Online Access: | http://eprints.nottingham.ac.uk/39144/ http://eprints.nottingham.ac.uk/39144/1/AshaPatel%20Thesis%202013.pdf |
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nottingham-39144 |
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eprints |
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nottingham-391442017-10-19T17:35:29Z http://eprints.nottingham.ac.uk/39144/ Chemically diverse polyacrylate and polyacrylamide surfaces for human cardiomyocyte culture and their effect on phenotype Patel, Asha K. Human pluripotent stem cell (hPSC) derived cardiomyocytes can provide robust in vitro models for pharmaceutical drug screening and modelling cardiac disease. To fully realise these potentials, hPSC-cardiomyocytes must be reproducibly cultured to a more mature state than has thus far been achieved. Defined and well controlled culture conditions underpin the ability to derive hPSC-cardiomyocytes of consistent quality. These include cell source, culture medium and substrate. This thesis is concerned with the latter; culture substrates are currently biological in nature creating inherent variability in culture conditions. There is limited knowledge on the interaction between cardiomyocytes and synthetic, non-biological substrates making rational design of materials impractical. To aid discovery of novel culture substrates, 115 polyacrylate and polyacrylamide substrates were microarrayed and investigated using a parallel screening approach. The use of polyacrylates and polyacrylamides as biomaterials has been demonstrated previously, including contact lenses, bone cements and hydrogels to support cells. The diverse chemistry exhibited across the range of polymers enables modelling of structure-activity relationships between substrate chemistry and cardiomyocyte behaviour. Cardiomyocytes derived from human embryonic stem cells (hESC) were cultured on the microarrays in the presence and absence of serum. Attachment density (nuclei count via DAPI staining) and cardiomyocyte spread (surface area using sarcomeric α-actinin immunostaining) on each substrate was acquired by automated fluorescence microscopy and image analysis software. From this primary screen, 70 % of polymers were found to support cardiomyocytes adhesion in serum conditioned arrays, BM34 (fufuryl methacrylate) supported the highest relative cell density (0.59 ±0.28) and also the largest cell size (1364 ±937 µm2), which was comparable to gelatin control. In serum free conditions, only 10 % of polymers supported cardiomyocyte attachment. The highest relative cell density was on AM38 (Dimethylamino-propyl acrylate) and largest cell size on BM49 (Tert-butylamino-ethyl methacrylate), 274 ±75 µm2, which was significantly lower than cell density and cell area (2019 ±596 µm2) on gelatin control. To investigate if synergy exists between polymers that enable adhesion in serum free conditions and those that support larger cell areas in serum conditioned arrays, 24 polymers were mixed pair-wise to form second generation microarrays comprising of 576 co-polymers. This diverse library enabled unique combinations of chemical moieties to be explored and co-polymers were found to have a greater proportion (74 %) that supported cardiomyocyte attachment in serum free conditions, largest average cell size was now 1089 ±260 µm2 on BM80/AM64 (Methacryloyloxy)ethyl acetoacetate/ Hexadecafluoro-9-(trifluoromethyl)decyl acrylate). Five co-polymers were chosen to perform more detailed characterisation of cardiomyocytes cultured on them for 5 and 25 days. Electrophysiological profiling and quantification of myofibril organisation identified co-polymers AD17/BM54 (Hexanediol ethoxylate diacrylate/ Ethoxyethyl methacrylate) and BM80/AD17 to be comparable to control gelatin. Partial least squares multivariate regression analysis correlated chemical species from the polymeric substrate, identified using time of flight secondary ion mass spectrometry, with cardiomyocyte response and identified moieties beneficial or detrimental for cardiomyocyte adhesion and cell area. This may aid rational design of tailor-made non-biological substrates for cell culture. In summary, the parallel screening of co-polymers of acrylates and acrylamides has been the first step in a discovery process of lead materials capable of progressing the culture of cardiomyocytes in more reproducible, economical and defined conditions. Only five substrates were analysed in detail, leaving a large library of co-polymers worthy of further investigation, including the physical properties of the polymers that need to be considered for practical use of the polymers in culture. 2014-07-08 Thesis (University of Nottingham only) NonPeerReviewed application/pdf en http://eprints.nottingham.ac.uk/39144/1/AshaPatel%20Thesis%202013.pdf Patel, Asha K. (2014) Chemically diverse polyacrylate and polyacrylamide surfaces for human cardiomyocyte culture and their effect on phenotype. PhD thesis, University of Nottingham. |
| repository_type |
Digital Repository |
| institution_category |
Local University |
| institution |
University of Nottingham Malaysia Campus |
| building |
Nottingham Research Data Repository |
| collection |
Online Access |
| language |
English |
| description |
Human pluripotent stem cell (hPSC) derived cardiomyocytes can provide robust in vitro models for pharmaceutical drug screening and modelling cardiac disease. To fully realise these potentials, hPSC-cardiomyocytes must be reproducibly cultured to a more mature state than has thus far been achieved. Defined and well controlled culture conditions underpin the ability to derive hPSC-cardiomyocytes of consistent quality. These include cell source, culture medium and substrate. This thesis is concerned with the latter; culture substrates are currently biological in nature creating inherent variability in culture conditions.
There is limited knowledge on the interaction between cardiomyocytes and synthetic, non-biological substrates making rational design of materials impractical. To aid discovery of novel culture substrates, 115 polyacrylate and polyacrylamide substrates were microarrayed and investigated using a parallel screening approach. The use of polyacrylates and polyacrylamides as biomaterials has been demonstrated previously, including contact lenses, bone cements and hydrogels to support cells. The diverse chemistry exhibited across the range of polymers enables modelling of structure-activity relationships between substrate chemistry and cardiomyocyte behaviour.
Cardiomyocytes derived from human embryonic stem cells (hESC) were cultured on the microarrays in the presence and absence of serum. Attachment density (nuclei count via DAPI staining) and cardiomyocyte spread (surface area using sarcomeric α-actinin immunostaining) on each substrate was acquired by automated fluorescence microscopy and image analysis software.
From this primary screen, 70 % of polymers were found to support cardiomyocytes adhesion in serum conditioned arrays, BM34 (fufuryl methacrylate) supported the highest relative cell density (0.59 ±0.28) and also the largest cell size (1364 ±937 µm2), which was comparable to gelatin control. In serum free conditions, only 10 % of polymers supported cardiomyocyte attachment. The highest relative cell density was on AM38 (Dimethylamino-propyl acrylate) and largest cell size on BM49 (Tert-butylamino-ethyl methacrylate), 274 ±75 µm2, which was significantly lower than cell density and cell area (2019 ±596 µm2) on gelatin control.
To investigate if synergy exists between polymers that enable adhesion in serum free conditions and those that support larger cell areas in serum conditioned arrays, 24 polymers were mixed pair-wise to form second generation microarrays comprising of 576 co-polymers. This diverse library enabled unique combinations of chemical moieties to be explored and co-polymers were found to have a greater proportion (74 %) that supported cardiomyocyte attachment in serum free conditions, largest average cell size was now 1089 ±260 µm2 on BM80/AM64 (Methacryloyloxy)ethyl acetoacetate/ Hexadecafluoro-9-(trifluoromethyl)decyl acrylate). Five co-polymers were chosen to perform more detailed characterisation of cardiomyocytes cultured on them for 5 and 25 days. Electrophysiological profiling and quantification of myofibril organisation identified co-polymers AD17/BM54 (Hexanediol ethoxylate diacrylate/ Ethoxyethyl methacrylate) and BM80/AD17 to be comparable to control gelatin.
Partial least squares multivariate regression analysis correlated chemical species from the polymeric substrate, identified using time of flight secondary ion mass spectrometry, with cardiomyocyte response and identified moieties beneficial or detrimental for cardiomyocyte adhesion and cell area. This may aid rational design of tailor-made non-biological substrates for cell culture.
In summary, the parallel screening of co-polymers of acrylates and acrylamides has been the first step in a discovery process of lead materials capable of progressing the culture of cardiomyocytes in more reproducible, economical and defined conditions. Only five substrates were analysed in detail, leaving a large library of co-polymers worthy of further investigation, including the physical properties of the polymers that need to be considered for practical use of the polymers in culture. |
| format |
Thesis (University of Nottingham only) |
| author |
Patel, Asha K. |
| spellingShingle |
Patel, Asha K. Chemically diverse polyacrylate and polyacrylamide surfaces for human cardiomyocyte culture and their effect on phenotype |
| author_facet |
Patel, Asha K. |
| author_sort |
Patel, Asha K. |
| title |
Chemically diverse polyacrylate and polyacrylamide surfaces for human cardiomyocyte culture and their effect on phenotype |
| title_short |
Chemically diverse polyacrylate and polyacrylamide surfaces for human cardiomyocyte culture and their effect on phenotype |
| title_full |
Chemically diverse polyacrylate and polyacrylamide surfaces for human cardiomyocyte culture and their effect on phenotype |
| title_fullStr |
Chemically diverse polyacrylate and polyacrylamide surfaces for human cardiomyocyte culture and their effect on phenotype |
| title_full_unstemmed |
Chemically diverse polyacrylate and polyacrylamide surfaces for human cardiomyocyte culture and their effect on phenotype |
| title_sort |
chemically diverse polyacrylate and polyacrylamide surfaces for human cardiomyocyte culture and their effect on phenotype |
| publishDate |
2014 |
| url |
http://eprints.nottingham.ac.uk/39144/ http://eprints.nottingham.ac.uk/39144/1/AshaPatel%20Thesis%202013.pdf |
| first_indexed |
2018-09-06T12:57:15Z |
| last_indexed |
2018-09-06T12:57:15Z |
| _version_ |
1610862963503136768 |