Towards Retinal Repair: Bioelectric Assessment of Retinal Pigment Epithelium in vitro and Electrode Materials for Retinal Implants

The aim of this thesis was to develop methods for future solutions to prevent eye diseases caused by the dysfunctions of retinal pigment epithelial (RPE) cells and to restore the vision of blind patients. On a cellular level, the degeneration of RPE cells is often the prime cause of eye diseases such as age-related macular degeneration and some forms of retinitis pigmentosa. RPE cell replacement therapy may provide new solutions for the prevention of eye diseases that lead to blindness. RPE cells differentiated from pluripotent stem cells provide a promising source for cell replacement therapy. However, the functionality of the differentiated cells is still not fully proven. One objective of this thesis was to provide solutions for testing the functionality of differentiated RPE cells. If blindness cannot be cured, artificial vision provided by retinal implant may be considered. The second objective of this thesis was to characterize the electrochemical properties of the different electrode materials used in retinal implants. The electrode materials used in retinal implants should be carefully considered in order to increase the resolution of the implant and to provide stable, safe, and biocompatible charge injection. All the methods used and developed in this thesis were based on bioelectrical phenomena. The electrochemical characterization of five different electrode materials used in retinal implants used electrical impedance spectroscopy (EIS) and cyclic voltammetry (CV) measurements. We considered the effect of electrode size and material on charge capacity and impedance. Atomic force microscopy (AFM) was used to study the surface properties of the studied electrodes. The testing of the materials was done using exactly the same measurement conditions and electrode producing methods to provide easily comparable data. In this thesis, the functionality of RPE cells differentiated from human embryonic stem cells (hESC-RPE) was studied with two different methods. EIS was used to compare the electrical properties between two different RPE cell lines (immortalized human RPE cell line (ARPE-19) and hESC-RPE). To our knowledge, EIS measurements of RPE cells have not been published before. EIS was also used to find out how the barrier properties of hESC-RPE cells differ when the cells are in different stages of maturity. In addition, we developed a method that could be used to study the functionality of hESC-RPE cells with in vitro electroretinography (ERG) measurements: Our hypothesis is that RPE cells enhance the ERG response of the mouse retina and enable longer culturing of the functional retina in vitro. Comparing the ERG responses of a mouse retina alone and of a mouse retina cultured together with hESC-RPE cells could reveal the functionality of hESC-RPE cells. The EIS measurements were in accordance with biological analyses. The hESC-RPE cells resembled morphologically mature RPE, and thus created high transepithelial resistance (TER) indicating high integrity and tight junction formation. The EIS measurements revealed that during the maturation the TER of the cell culture increases, peak phase diagram shifts to lower frequencies, and the capacitance of the epithelium increases. Permeability measurements verified that EIS measurements reveal the tight junction failures and integrity decrease caused by calcium chelation. With the developed setup we were able to measure ERG responses from both the co-culture of retina and RPE and the retina cultured alone. However, due to limited sample size and possibly due to short co-culture time in our culture setup as yet we were not able to prove the hypothesis by showing that RPE cells would enhance the ERG response of the retina in vitro. Both the retina cultured alone and the co-culture responded to light stimulus after one day of culturing. CV and EIS measurements of different electrodes showed that iridium-black (Ir-b) and platinum-black (Pt-b) electrodes were superior, i.e. they had higher charge injection capacity and lower impedance when compared to other tested materials (gold (Au), titaniumnitrate (TiN), titanium (Ti)). Based on our findings we can conclude that novel biocompatible electrode materials that have the potential to be used in implantation are available. In the same way as in this thesis, the electrochemical testing of electrode materials should be done using similar testing methods for every material to enable easy comparison of the results between different materials. At the moment, cell replacement therapy and the use of RPE cells is seriously considered as a choice for eye disease treatment. Our results suggest that EIS is useful when evaluating the overall maturity, integrity, and functionality of the RPE cell culture. In forthcoming cell transplantation therapies, EIS could provide a means to test the validity of stem cell-derived RPE non-invasively and aseptically before implantation. Our initial tests show that studies to test the ability of RPE cells to rescue the photoreceptors in a mouse model by testing ERG responses in vitro should be continued. Even though our results did not produce conclusive evidence, the co-culture of the retina and hESC-RPE cells may be a useful in vitro model for investigating the RPE cell replacement therapy and possible drug releasing materials for the retina

Human Embryonic Stem Cell-Derived Retinal Pigment Epithelium-Role in Dead Cell Clearance and Inflammation

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Geenimutaatiot ja niihin liittyvät mitokondriohäiriöt : diagnostiikka ja ihmisen indusoitujen pluripotenttien kantasolujen soveltaminen tautien syntymekanismien selvittämisessä

Mitochondrial diseases are generally caused by genetic variants that may affect cell function during the process of energy generation: right from the start of protein translocation to the fatty acid degradation by beta-oxidation (β-oxidation). The main objective of this PhD thesis is to study genetic variants that cause mitochondrial diseases and also to understand the disease pathogenesis of a known disease using the induced pluripotent stem cell (iPSC) method, a revolutionary approach in regenerative medicine. In the first study, we carried out a long-term follow up of six metabolic diseased patients and subsequently we performed a carrier frequency study of the identified carnitine palmitoyl transferase 1A (CPT1A) gene variant in the Finnish population. We identified a novel homozygous variant c.1364A>C (p.Lys455Thr) in exon 12 of the CPT1A gene. No carriers of the variant c.1364A>C were detected upon minisequencing of 150 control samples but the allele frequency of CPT1A variant in global population is 0.0002142 (ExAC Browser) whereas in the Finnish population (6614 allele number) the frequency is 0.001966. The identified variant was predicted to cause improper folding of the CPT1A protein, which leads to its degradation. All patients were treated with a high-carbohydrate and a low fat diet. In the second study, we focused on the human DnaJ (Hsp40 homolog) subfamily C, member 19 (DNAJC19) deficiency. Our studies showed that it causes early onset dilated cardiomyopathy syndrome (DCMA). This is the first report of a genetic defect in the mitochondrial protein, DNAJC19, outside of the Canadian Dariusleut Hutterite population. This defect is characterized by an unusual aetiology for an early onset recessively inherited dilated cardiomyopathy that is associated with ataxia and male genital anomalies. Sequencing of the human DNAJC19 gene revealed a homozygous single nucleotide (A) deletion in exon 6 that cause a frameshift and lead to the premature termination of the protein. In the third study, the pathogenesis of retinopathy in long-chain acyl-CoA dehydrogenase deficiency (LCHADD) was studied using iPSC technology. Retinopathy is an unusual manifestation of LCHADD, as mitochondrial fatty acid β-oxidation (FAβO) has not been considered to play a major role in the metabolism of the retina. Among all defects of mitochondrial FAβO, only long-chain acyl-CoA dehydrogenase (LCHAD) and mitochondrial trifunctional protein (TFP) deficiencies have developed pigmentary retinopathy and peripheral neuropathy. We elucidated how a genetic variant in the FAβO cycle can disrupt the retinal pigment epithelium (RPE) that can eventually lead to blindness. In addition, we developed a new in vitro cell model; iPSC clones were generated from LCHADD patient fibroblasts and further differentiated into RPE cells. Several changes were observed in patient RPE cells such as decreased cell size, lower pigmentation and irregular pattern of morphology. Electron microscopy analysis showed an accumulation of a few melanosomes, more melanolysosomes, and large sized lipid droplets in patient RPE cells. Furthermore, increased levels of triglycerides in patient RPE cells were observed upon mass spectrometric analysis. We concluded that all these changes had contributed to the disruption of the RPE layer that leads to blindness in LCHAD deficiency patients. Finally, the research done for this thesis succeeded in identifying novel variants in CPT1A and DNAJC19 genes in Finnish patients. Our long-term follow up studies on CPT1A deficiency can help patients in better diagnosis, which further helps clinicians to identify the genetic cause. We also found a novel phenotype with DNAJC19 deficiency. Further we established the groundwork to understand the pathogenesis of retinopathy in LCHADD patients using an advanced method that helps to study in depth pathogenesis mechanism.Monien ihmisten päivittäiseen ruokavalioon kuuluu rasvaa sisältäviä elintarvikkeita, kuten juustoa, maitoa, voita ja öljyä. Rasva imeytyy verenkiertoon ja kulkeutuu eri ruumiinosiin ja kudoksiin tuottamaan energiaa. Kun kaloreiden tarve on täytetty, ylimääräinen rasva varastoituu triglyserideiksi rasvakudokseen. Glukoosi on tärkein energianlähde, mutta kun glukoositasot ovat alhaiset (esimerkiksi paaston tai sairauden aikana), varastoidut rasvat muuntuvat energiaksi rasvahappojen beetaoksidaatioksi kutsutussa prosessissa, joka tapahtuu mitokondrioina tunnetuissa soluelimissä. Mitokondriot ovat solujen voimaloita, jotka muuntavat energian adenosiinitrifosfaatiksi (ATP) kutsutuksi polttoaineeksi. Geenit puolestaan ovat sukupolvelta toiselle periytyvää DNA-ainesta, joka välittyy lapsille vanhempien kautta. Jos geenin muodostuessa tapahtuu mutaatio, mekanismi häiriintyy ja aiheuttaa sairauden. Tämä opinnäyte tutkii kolmea suomalaisväestössä esiintyvää geenivirhettä, jotka liittyvät rasvahappojen beetaoksidaatioon. Ne vaikuttavat pitkäketjuisten rasvahappojen hajottamiseen ja proteiinien kuljettamiseen mitokondrioihin. Kaksi ensimmäistä geenivirhettä ovat karnitiinipalmityylitransferaasi 1 A (CPT1A) ja DNAJC19. Nämä geenivirheet todetaan yleensä vastasyntyneillä ja taaperoikäisillä lapsilla (toiseen ikävuoteen mennessä), ja niihin liittyviä oireita ovat esimerkiksi alhainen verenpaine, oksentelu ja kohtaukset. Biokemiallisten veri- ja virtsakokeiden jälkeen potilailta otettiin veri- ja ihonäytteitä (lupa saatu) DNA:n eristämistä ja geenivirheiden toteamista varten. Suomalaisväestössä havaittu mutaatio on c. 1364A>C (p.Lys455Thr) CPT1A-geenin eksonissa 12. Diagnoosin jälkeen geeniä kantavat potilaat noudattivat vähärasvaista ruokavaliota, joka paransi heidän metabolisia arvojaan. DNAJC19 on ensimmäinen raportoitu mitokondrion proteiinien geenivirhe kanadalaisen Dariusleut-hutteriittiväestön ulkopuolella. Se aiheuttaa varhain puhkeavan dilatoivan kardiomyopatian ja ataksian. Yhden nukleotidin puute (A) johtaa proteiinin terminaatioon. Kolmas tutkittu geeni liittyy pitkäketjuisten rasvahappojen asyyli-CoA-dehydrogenaasi (LCHAD) -entsyymiin, joka osallistuu rasvahappojen beetaoksidaatioon auttamalla pitkäketjuisten rasvahappojen hajottamista. LCHAD:n puutos aiheuttaa sokeuteen johtavaa retinopatiaa. LCHAD-potilaiden retinopatian tutkimiseen käytettiin kantasolutekniikkaa, jolla tuotettiin ihmisen indusoituja pluripotentteja kantasoluja (hiPSC) potilaiden ihosoluista käyttämällä neljää merkittävää transkriptiotekijää. Kyseiset hiPSC:t erilaistetaan edelleen verkkokalvon pigmenttiepiteeleiksi (RPE), jotka ovat tärkeitä näkökyvylle. Morfologiset aineistot, elektronimikroskopia ja massaspektrometria paljastivat lipidikertymiä potilaiden RPE-soluissa. Tämä viittaa siihen, että lipidikertymät saattavat vaikuttaa LCHAD-puutosta sairastavien potilaiden retinopatiaan

Elektroniikan pääteaste nopeisiin biologisiin in vitro mittauksiin.

Epithelium forms tight membranes that efficiently take part in secretion and absorption between the two lining tissues. A tight membrane of this nature has a low DC conductivity and this is generally used to assess the integrity of cultured epithelium cell layers. Recent studies have shown that impedance spectroscopy gives more information about the electrophysiological structure of the cells. Collaborative research by Department of Biomedical Engineering at Tampere University of Technology and stem cell research group at University of Tampere has shown that electrochemical impedance spectroscopy is useful in assessing the maturity and functionality of differentiated retinal pigment epithelium (RPE) cells. However the time expenditure of the traditional frequency sweeping method is a poor candidate for drug permeability or multichannel studies where several frequency responses have to be measured within a short time. The aim of this Thesis was to develop the front end electronics for fast impedance spectroscopy measurement employing inverse-repeat binary sequence as the broadband excitation signal. Also the DC potential across the cell membrane was to be measured with the device. The developed device was first tested using a custom built test box and plastic film as artificial membrane. In addition several electrode materials were used to study the observed polarization impedances. Further testing with differentiated RPE cell layers was done using the Ussing chamber and the well plate setups that are commonly used in cell culturing studies. All the measured frequency responses were referenced with a commercial device widely used in epithelium research. The observed measurement differences between the device and the reference were largely caused by the load dependent output current of the device and by the electrode polarization taking place at the voltage measurement electrodes. Due to the input current error the relative difference of the measured impedance levels was typically from 5 % to 10 % with load impedances larger than 700 ohms. With lower load impedances the measured relative difference increased rapidly. A method to compensate for the input current error is presented in this thesis. DC potential measurements with the device were not successful as the electrodes used had very high offset voltages. The frequency responses measured with the device give a good measure of the capacitance present in the cell layer. Capacitance of the cell layer can be used to assess the maturity of the cell layer and for such purpose the device suits well. For impedance level measurements the device has a relatively large error margin and further research needs to be done to improve the accuracy and to eliminate the DC current flow. In addition the accuracy of the measurement system would improve by dividing the input stage between the DC potential and frequency response measurements. Also more carefully designed electrodes would help to control the electrode offset voltages.Epiteelisolut muodostavat tiivisliitoksia, jotka ovat tärkeässä roolissa kudosten välillä tapahtuvassa erityksessä sekä absorptiossa. Tiivisliitoksen tasavirtajohtavuus on heikko ja tätä ominaisuutta hyödynnetään yleisesti viljeltyjen epiteelisolukerrosten tiiviyden sähköisessä tarkastelussa. Tutkimukset ovat kuitenkin osoittaneet, että impedanssispektroskopia antaa enemmän tietoa solujen elektrofysiologisesta rakenteesta kuin yksinkertainen resistanssimittaus. Tampereen Teknillisen Yliopiston Biolääketieteen laitos sekä Tampereen Yliopiston kantasolututkimusryhmä ovat yhteistyössä osoittaneet elektrokemiallisen impedanssispektroskopian soveltuvan kantasoluista erikoistettujen retinan pigmenttiepiteelisolujen (RPE) kypsyyden ja toiminnallisuuden arviointiin. Perinteiset taajuuspyyhkäisyä hyödyntävät taajuusvasteanalysaattorit soveltuvat kuitenkin hitaudeltaan heikosti tutkimuksiin, joissa mittauskohteessa tapahtuu nopeita muutoksia tai missä useita taajuusvasteita mitataan lyhyellä aikavälillä. Tämän diplomityön tavoitteena oli kehittää pääteasteen elektroniikka mittausjärjestelmälle, joka hyödyntää laajakaistaista binäärijaksoa herätesignaalina ja mahdollistaa näin huomattavasti nopeamman impedanssispektroskopian. Kehitettävän laitteen tuli myös mitata solukerroksen yli oleva DC potentiaali. Diplomityössä kehitettyä laitetta testattiin aluksi in vitro mittauksia varten kehitetyllä testijärjestelmällä, jossa solukerrosta jäljiteltiin ohuella muovikalvolla. Näissä mittauksissa testattiin myös eri elektrodimateriaalien vaikutus havaittuun polarisaatioimpedanssiin. Viljeltyjen RPE solujen taajuusvasteita mitattiin työssä käyttäen sekä Ussingin kammio- että kuoppalevymittausasetelmia. Laitteella mitattujen taajuusvasteiden hyvyyttä arvioitiin vertaamalla tuloksia kaupallisella taajuusvasteanalysaattorilla mitattuihin vasteisiin. Kehitetyllä laitteella mitattujen vasteiden ero analysaattorilla mitattuihin johtui suurilta osin herätevirran riippuvuudesta kuormasta sekä jännitemittauselektrodien polarisaatioimpedansseista. Yli 700 ohmin kuormilla herätevirrasta aiheutuva virhe oli tyypillisesti 5% – 10 %, kun taas matalimmilla kuormilla virhe kasvoi nopeasti. Tämän virheen kompensoimiseksi on kuitenkin esitetty metodi tässä diplomityössä. Epiteelisolukerroksen yli olevaa DC jännitettä ei onnistuttu mittaamaan johtuen käytettyjen elektrodien korkeista offset-jännitteistä. Tässä diplomityössä kehitetyllä laitteella mitatut taajuusvasteet noudattavat hyvin analysaattorilla mitattujen vasteiden käyrämuotoja ja laite soveltuu täten solukerroksen kapasitanssin arviointiin. Solukerroksen kapasitanssia voidaan käyttää apuna solujen kypsyyden arvioinnissa. Laitteella mitatut impedanssitasot eroavat kuitenkin analysaattorilla mitatuista ja jatkokehitys DC virtojen eliminoimiseksi sekä elektrodien erisuuruisten offset-jännitteiden kompensoimiseksi on suositeltavaa

Fabrication and properties of woven structures for biomedical applications

The purpose of this work was to manufacture a dense structure with small pores for biomedical studies. The structures are made of fine multifilament yarn by weaving. The aim was to get the yarn densities as high as possible, which would decrease the pore size of the structure. Nine samples were woven by using different weave patterns and different warp densities. Pore sizes and evenness of the structures were compared by microscope imaging. Mass per unit area, thickness and tensile strength of the samples were compared. Two of the best structures were woven with the help of paraffin oil as a lubricant to get even higher warp densities and to reduce the friction between the yarns, thus preventing them from breaking. Contact angles of the same samples were measured to get a better view of the permeability and porosity. The best weave patterns had several evenly distributed interlacing points. Best results were achieved by using the paraffin oil; there were only few through-going openings. They were only a few micrometres in size and they were distributed randomly around interlacing points.Työn tarkoituksena oli valmistaa biolääketieteen tutkimuskäyttöön tiivis pienihuokoinen alusta kutomalla hienoa multifilamenttilankaa. Työssä pyrittiin saamaan kudotun rakenteen lankatiheydet mahdollisimman suuriksi, mikä pienentäisi siten rakenteen huokoskokoa. Rakenteita kudottiin käyttämällä yhdeksää erilaista sidosta eri loimitiheyksillä ja mahdollisimman suurella kudetiheydellä. Näytteiden huokoskokoa ja tasaisuutta vertailtiin käyttäen apuna mikroskooppia. Lisäksi näytteiden neliömassat, paksuudet ja vetolujuudet olivat vertailtavina. Kaksi parhainta näytettä näistä yhdeksästä kudottiin myös käyttäen apuna parafiiniöljyä voiteluaineena, jotta loimitiheyksiä saatiin vielä suuremmiksi. Parafiiniöljyä käytettiin kudonnan aikana vähentämään loimilankojen välistä kitkaa ja siten estämään niiden katkeamista. Myös kontaktikulmat mitattiin näistä kahdesta parhaasta näytteestä, mikä kertoi enemmän niiden läpäisevyydestä ja huokoisuudesta. Parhaimmat sidokset olivat nimenomaan ne, joissa sidospisteitä oli tiheässä ja ne olivat tasaisesti jakautuneet kankaan pinnalle. Voiteluainetta apuna käyttämällä saatiin parhaat tulokset; läpimeneviä huokosia oli hyvin vähän, ne olivat jakaantuneet satunnaisten sidospisteiden ympäristöön ja ne olivat kooltaan vain muutamia mikrometrejä

Development and clinical application of impedance pneumography technique

Assessment of the lung function is essential in the diagnosis and management of respiratory disease such as asthma. However, conventional spirometry requires difficult manoeuvres from the subject and is thus unsuitable for young children and infants. This renders the diagnosis of childhood asthma often qualitative, time-consuming and clinically challenging. However, information relating to the lung function can be derived from restful tidal breathing (TB) as well. Traditionally TB has been recorded in short intervals in laboratory conditions with obtrusive instrumentation using a face mask or a mouth piece. The principal aim of this thesis was to develop a noninvasive and convenient, yet highly accurate method for recording TB over extended time periods for clinical purposes, especially in young children. The measurement methodology developed within this thesis is based on impedance pneumography (IP), where breathing is recorded through the respiratory variations of the electrical impedance of the thorax. This is established by placing four skin electrodes on the upper body and connecting them to a recording device. The main focus was in ensuring the accuracy of the IP-derived tidal flow recording as compared to direct measurement from the mouth. This was established by attenuating the distortive cardiac oscillations (CGO) of the impedance signal and by optimising the locations of the skin electrodes. The complete method was then validated in healthy adults during respiratory loading (n=17) and in preschool children with wheezing disorder (n=20). The CGO attenuation was realised through an ensemble averaging based signal processing algorithm. The algorithm takes into account the respiratory modulation of the CGO waveform thus enabling efficient CGO attenuation while preserving the respiratory component of the signal unchanged. The newly proposed electrode configuration provides consistently more linear impedance to lung volume ratio than those previously established in the literature. The complete method integrating these developments provided highly accurate TB flow signal during normal and altered respiratory mechanics (loading) in adults and during induced bronchoconstriction in young children. It may be concluded that in this thesis significant improvements were realised with the IP technique. These improvements were experimentally validated in two studies and the integrated system was found to consistently provide an accurate respiratory flow signal. The method may have clinical implications for the diagnosis of respiratory diseases especially in non-cooperative subjects, such as young children