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Difficulties in obtaining and maintaining the desired level of the critical quality attributes (CQAs) of therapeutic proteins as well as the pace of the development are major challenges of current biopharmaceutical development. Therapeutic proteins, both innovative and biosimilars, are mostly glycosylated. Glycans directly influence the stability, potency, plasma half-life, immunogenicity, and effector functions of the therapeutic. Hence, glycosylation is widely recognized as a process-dependent CQA of therapeutic glycoproteins. Due to the typically high heterogeneity of glycoforms attached to the proteins, control of glycosylation represents one of the most challenging aspects of biopharmaceutical development. Here, we explored a new glycoengineering approach in therapeutic glycoproteins development, which enabled us to achieve the targeted glycoprofile of the Fc-fusion protein in a fast manner. Coupling CRISPRi technology with lectin-FACS sorting enabled downregulation of the endogenous gene involved in fucosylation and further enrichment of CHO cells producing Fc-fusion proteins with reduced fucosylation levels. Enrichment of cells with targeted glycoprofile can lead to time-optimized clone screening and speed up cell line development. Moreover, the presented approach allows isolation of clones with varying levels of fucosylation, which makes it applicable to a broad range of glycoproteins differing in target fucosylation level.

V zadnjih nekaj desetletjih so se za gojenje ovarijskih celic kitajskega hrčka (celice CHO) na laboratorijskem nivoju za potrebe razvoja uporabljale rastne plastenke (SF). Zaradi vse večjih potreb po (podobnih) bioloških zdravilih, biofarmacevtska industrija teži k čim večjim izkoristkom proizvodnje in čim krajšim časovnicam razvoja omenjenih zdravil. To je razlog, da SF niso več ustrezni za gojenje celic CHO, saj ne omogočajo visoke zmogljivosti dela. V praksi neučinkovite SF sedaj nadomeščajo plošče 24DW, ki so zelo primerne za avtomatizacijo in robotizacijo postopkov v celičnem laboratoriju. S ploščami 24DW dosegamo boljše izkoristke v smislu časa, števila proizvedenih celičnih klonov, obsega dela, porabljenih surovin in financ. Še boljše izkoristke in višje zmogljivosti laboratorija nam omogočajo plošče 96DW. Z avtomatiziranimi postopki in uporabo robotskih pipetirnih sistemov v 96DW gojimo več tisoč klonov hkrati brez povečanja obsega dela. Z njimi izvedemo učinkovitejše presejanje klonov ter tako hitreje pridobimo visoko producirajoče celične linije. Pri razvoju bioprocesa se večinoma uporablja miniaturizirane bioreaktorske sisteme (npr. Ambr 15), ki omogočajo gojenje do 48 celičnih linij hkrati pri skrbno kontroliranih in uravnavanih pogojih. Sistem Ambr 15 zahteva dokaj visok finančni vložek na izveden bioproces, a je visoko zmogljiv in avtomatiziran ter odlično posnema obnašanje celične linije v velikih bioreaktorjih. V sklopu magistrske naloge smo želeli ovrednotiti gojenje celic CHO v novih, učinkovitejših platformah (24DW, 96DW) glede na uveljavljene SF. Zaradi izrazito višjih časovnih, materialnih in finančnih izkoristkov novejših platform smo želeli preveriti, ali bi slednje lahko nadomestile neučinkovite SF pri gojenju celic CHO za potrebe razvoja produkcijskih celičnih linij. Prav tako smo želeli preveriti, kako platforme v zgodnjem razvoju (SF, 24DW, 96DW) napovejo dogajanje v Ambr 15. Parametri, ki smo jih tekom magistrske naloge vrednotili, so bili: število živih celic, viabilnost ter celična produktivnost (titer). V analize smo vključili tako meritve, pridobljene med 14-dnevnimi šaržami z dohranjevanjem kot tudi meritve med 6-dnevnimi šaržami brez dohranjevanja. Primerjani parametri v 24DW in 96DW dobro korelirajo z obstoječimi SF (ρ > 0,87 za vrednosti titra). Slabše, vendar vseeno zadovoljive korelacije smo dobili med meritvami parametrov v sistemu Ambr 15 in ostalimi platformami (ρ > 0,63 za vrednosti titra). Hkrati smo z eksperimenti pokazali, da lahko s 6-dnevno šaržo brez dohranjevanja dobro napovemo dogajanje po koncu 14-dnevne šarže z dohranjevanjem. Vse bolj se zavedamo tudi pomembnosti poznavanja, modeliranja in kontrole N-glikozilacije monoklonskih protiteles oz. Fc-fuzijskih proteinov. Glikanski profili namreč vplivajo na aktivnost biofarmacevtika, na njegove farmakokinetične in farmakodinamične lastnosti ter na varnost njegove uporabe. V sklopu magistrske naloge smo zato s pomočjo interferometrije z biološkimi plastmi (analiza BLI) ovrednotili tudi glikanske profile proteina v različnih platformah. Ugotovili smo, da se glikanski profil proteina ohrani ne glede na uporabljeno platformo. Dobro ohranjenost glikanskega profila smo pokazali tudi med 14-dnevno šaržo z dohranjevanjem in 6-dnevno šaržo brez dohranjevanja. In the last few decades shake flasks (SF) were traditionally used for small scale Chinese hamster ovary cell (CHO cell) cultivation in development of biopharmaceuticals. The need for novel biopharmaceuticals is increasing and biopharmaceutical industry is striving for high efficiency of production processes and shorter timelines for drug development. Due to above mentioned reasons conventional SFs are not sufficient anymore as they do not provide high-throughput workflow. In recent years SFs were replaced by high-throughput 24DW plates that are suitable for automation and robotization. By using 24DW we are able to reach higher efficiency regarding costs, time, number of produced clones and workload. Even better efficiencies are reached when using 96DW plates. With highly automated and robotized laboratory procedures we can handle thousands of clones in 96DW plates in parallel. Clone screening in this platform is very effective and the final production clone is selected quicker. In up-stream process development the use of miniaturized bio-reactor systems (e.g. Ambr 15) is increasing. This platform provides cultivation of 48 different clones simultaneously in highly controlled and regulated conditions. It requires decently high financial input however, it is mainly automated, high-throughput system that perfectly mimics cell line behavior in large-scale bioreactors. In the present master thesis we evaluated cultivation of CHO cells in novel, high-throughput platforms (24DW, 96DW) and traditional SFs. Due to increasing development costs, need for higher resource and time efficiency we investigated whether novel platforms could replace the insufficient SFs while culturing CHO cells on laboratory scale. We also examined whether platforms in early development could mimic the bioprocess in Ambr 15. We analyzed different parameters such as viable cell density, viability and cell productivity (titer). In the evaluation of platforms we included data gathered during 14-day fed-batch as well as 6-day simple batch experiments. We observed very good correlation between 24DW, 96DW and conventional SFs cultivations (ρ > 0,87 for titer values). Correlations were slightly worse between Ambr 15 and other platforms (ρ > 0,63 for titer values). We showed that results from 6-day simple batch cultures predict the parameters at the end of 14-day fed-bach cultures in a fairly good manner. N-glycosylation of monoclonal antibodies and fusion proteins is particularly important nowadays, as it affects protein’s effector function, pharmacokinetics and pharmacodynamics as well as its safety in patient use. In this master thesis we evaluated whether the glycoprofile is preserved during CHO cell cultivation in different novel and already established platforms. Glycoprofiles were determined using bio-layer interferometry (BLI) measurements. We showed the glycan structure on expressed protein is mainly preserved regardless of the platform or cultivation regime used in experiments.

Kapljična digitalna verižna reakcija s polimerazo (ddPCR) je novejša oblika digitalne verižne reakcije s polimerazo (dPCR). Metoda temelji na tvorbi približno 20.000 kapljic, ki nastanejo v emulziji voda v olju (V/O), verižni pomnožitvi s polimerazo in končni absolutni kvantifikaciji kapljic s prisotnim pomnoženim produktom. Metoda je uporabna na številnih področjih, v okviru magistrske naloge pa smo želeli preveriti, če jo lahko uporabljamo tudi za razvoj rekombinantnih bioloških učinkovin v biofarmacevtski industriji. Pri izbiri ustreznih klonov igra ključno vlogo karakterizacija števila kopij, integritete in stabilnosti transgena. V podjetju Lek d.d. v ta namen uporabljajo verižno reakcijo s polimerazo v realnem času (qPCR) in metodo prenosa po Southernu, ki pa ju želijo nadomestiti z novejšo metodo – ddPCR. Metodo ddPCR smo najprej optimizirali in nato preverili njeno uporabnost za genetsko karakterizacijo klonov ovarijskih celic kitajskega hrčka (CHO), uporabljenih v proizvodnji rekombinantnih proteinov, zlasti monoklonskih protiteles. Preverili smo analitske parametre ddPCR pri določanju števila kopij in integritete vstavljenih transgenov, izražanja lahke in težke verige protiteles v celičnih linijah in možnost uporabe ddPCR za kvantifikacijo rezidualne DNA v različnih stopnjah čiščenja zdravilne učinkovine. Določili smo široko dinamično območje, ki ga pokriva ddPCR tako za določanje koncentracije DNA (4 – 8000 kopij/μL), kot tudi mRNA (8 – 20.000 kopij/μL). Gre za visoko občutljivo metodo, saj je limita detekcije 1,5 – 3 kopije tarčnega gena/μL pri vzorcih DNA, ter 1,2 kopij/μL pri RNA vzorcih. Variabilnost metode pri kvantifikaciji DNA se giblje med 10 in 15 % in je nekoliko višja pri uporabi barvila EvaGreen v primerjavi s hidrolizirajočimi sondami. DdPCR smo primerjali z uveljavljenimi metodami in ugotovili, da je metoda primerna za določanje števila kopij in izražanja transgenov ter rezidualne DNA. Pri preverjanju integritete vstavljenih transgenov je ddPCR izkazala 80 % občutljivost, 88 % specifičnost in 82 % točnost glede na metodo prenosa po Southernu. Pri določanju stabilnosti vstavljenih transgenov je ddPCR izkazala 40 % občutljivost, 100% specifičnost ter 70 % točnost glede na prenos po Southernu. V razvoju bioloških zdravil je metoda ddPCR primerna za preverjanje integritete transgenov in določanje števila kopij pri presejanju klonov, saj je hitrejša in v primeru določanja števila kopij tudi točnejša. Za uporabo pri določanju stabilnosti pa bi ddPCR potrebovala še nadaljnjo optimizacijo. Droplet digital polymerase chain reaction (ddPCR) is a new form of digital polymerase chain reaction (dPCR). The method is based on the formation of about 20,000 droplets formed in a water-oil emulsion, a polymerase chain reaction and final absolute quantification of droplets with a multiplied product. Although the method is applicable in many fields, the scope of the master's thesis was to check if it can also be used in the development of biological substances in the biopharmaceutical industry. For the selection of suitable clones, characterization of copy number, integrity and stability of the transgene plays an important role. For this purpose, the pharmaceutical company Lek d.d. uses the Southern blot and real-time polymerase chain reaction (qPCR), which is sought to be replaced with the newer ddPCR method. The ddPCR method was first optimised and then tested for its suitability for the genetic characterization of Chinese Hamster Ovary (CHO) clones, used for the production of recombinant proteins - mostly monoclonal antibodies. We tested the analytical parameters of ddPCR for determination of copy number and integrity of inserted transgenes, expression of the light and heavy chain of the antibody in cell lines and the possibility of using ddPCR to quantify residual DNA at different stages of drug purification. DdPCR covers a wide dynamic range of concentration determination for both DNA (4 – 8000 copies/μL) and mRNA samples (8 – 20,000 copies/μL). This is a highly sensitive method, since the detection limit is 1.5 – 3 copies of the target gene/μL in the case of DNA samples and 1.2 copies/μL in the case of RNA samples. The variability of the method for DNA quantification is between 10 and 15% and is slightly higher for the EvaGreen dye compared to TaqMan probes. We compared ddPCR with established methods and demonstrated that ddPCR is a suitable method for determination of transgene copy number, transgene expression and residual DNA concentration. For determination of integrity of inserted transgenes, ddPCR displays a 80% sensitivity, 88% specificity and 82% accuracy compared to the Southern blot method. For the determination of stability of inserted transgenes ddPCR showed a 40% sensitivity, 100% specificity, and 70% accuracy, compared to Southern blot. Thus ddPCR represents a suitable method for determination of transgene integrity and copy number during the clone screening process, as it is faster and in the case of copy number determination also more accurate. To be used for transgene stability determination, ddPCR needs further optimisation.

Over the past 30 years, Chinese hamster ovary (CHO) cells have become a major tool for recombinant protein production, mainly for monoclonal antibodies (mAb) and other biologics. These drugs are widely used to treat cancer, autoimmune disorders and many other diseases, which were considered as untreatable just a few years ago. CHO cells are mammalian cells, which synthesize recombinant products in a form that is suitable for clinical trials and therapies. Nowadays most bioprocesses are fed-batch processes where chemically defined solutions are used to supplement substrates. Such solutions consist of glucose, amino acids, microelements, etc. Despite the addition of the aforementioned solutions during bioprocessing, some of the amino acids are still depleted. In preliminary processes we observed the depletion of asparagine, aspartate, histidine, tryptophan, phenylalanine, glutamate, serine, threonine, glycine, cysteine and lysine. With the adaptation of an already existing feed and the usage of newly developed feeds we were able to increase cell viability and mAb production. These observations showed the importance of understanding the metabolism of CHO cells and the availability of amino acids in the cell culture. Celice ovarijev kitajskega hrčka (CHO) so v zadnjih letih postale vodilni ekspresijski sistem na področju razvoja ter proizvodnje rekombinantnih proteinov. Monoklonska protitelesa in ostala novodobna zdravila so v redni uporabi za terapijo rakavih, avtoimunskih in drugih bolezni, ki so še pred kratkim veljale za neozdravljive. Celice CHO so zmožne sinteze rekombinantnih proteinov v obliki, primerni za uporabo tako v kliničnih raziskavah kot pozneje v terapijah. V razvoju bioloških zdravil se večinoma uporablja šaržni bioproces z dohranjevanjem, kjer kemijsko definirana gojišča in hranila vsebujejo substrate, kot so glukoza, aminokisline, mikroelemente in druge komponente. Kljub temu, da hranila vsebujejo večino aminokislin, se mnogokrat po končanem bioprocesu izkaže, da jih celice porabijo. V preliminarnih poskusih smo opazili pomanjkanje asparagina, aspartata, histidina, triptofana, fenilalanina, glutamata, serina, treonina, glicina, cisteina in lizina. Z adaptacijo obstoječih in uporabo novih hranil smo povečali celično viabilnost in sintezo monoklonskega protitelesa. S tem smo pokazali, da imata lahko poznavanje celičnega metabolizma in prisotnost aminokislin ključno vlogo pri izboljšanju učinkovitosti celic.

Industrial production of recombinant proteins in CHO cells commonly uses vectors possessing strong viral promoters. Though this results in high productivity, it is accompanied by cellular stress and economic loss. In this thesis, the viral CMV and four endogenous CHO promoters (β-Actin, RAD52, HSP90, ST6GAL1) driving different reporter genes were used. Several TFOs were designed for each promoter. TFOs can affect expression of a reporter gene by binding into the promoter region. The activity of each promoter was observed with the luciferase assay and later with flow cytometry, when a different reporter gene was used. The next step included the use of TFOs, first preincubated with the promoter containing plasmid pre-transfection, then interacting with the promoter by being endogenously transcribed from a separate plasmid. We have also observed the TFO’s effect on an endogenous ST6GAL1 promoter in two CHO cell lines and monitored triplex formation by using FRET-qPCR. Some TFOs decreased promoter activity, which was an expected form of action. Yet in some cases, an increase in promoter activity was observed. The molecular structure of the TFOs and their sequence-specific placement in the promoter enables interaction with different TFs, prevention of PIC assembly and even altering the chromatin state. If the role of the TFOs would be elucidated and predictable, they could become an important metabolic engineering switch in recombinant protein production. Industrijska proizvodnja rekombinantnih proteinov v celicah CHO pogosto posega po vektorjih z močnimi virusnimi promotorji. Njihova uporaba se odraža v visoki produktivnosti, ki pa jo hkrati spremlja celični stres in ekonomske izgube. V tej nalogi smo uporabili virusni promotor CMV ter štiri endogene promotorje CHO (β-aktin, RAD52, HSP90, ST6GAL1), ki so vodili različne poročevalske gene. Za vsakega izmed promotorjev smo načrtovali TFO, ki lahko prek vezave v promotor uravnavajo prepisovanje poročevalskega gena. Aktivnost promotorjev smo opazovali s pomočjo luciferaznega testa. Ko smo uporabili drug poročevalski gen, pa še s pretočno citometrijo. Naslednji korak je bil uporaba posameznega TFO, predinkubiranega s plazmidom, ki je vseboval posamezen promotor. Kasneje smo celice CHO kotransfecirali s plazmidom, ki je vseboval posamezen promotor ter hkrati s plazmidom, ki je vseboval zapis za posamezen TFO. Preizkusili smo tudi odziv endogenega promotorja ST6GAL1 v dveh celičnih linijah CHO ter spremljali tvorbo tripleksa s pomočjo FRET-qPCR. Nekateri TFO so aktivnost promotorja znižali, kar je bilo pričakovano. Presenetljivo pa se je v nekaterih primerih izražanje gena povišalo. Struktura TFO in njihova zaporedno-specifična vezava v promotor omogoča interakcijo TFO z različnimi TF. TFO lahko preprečijo tvorbo PIC in celo spremenijo stanje kromatina. Če bi bila njihova vloga jasna in predvidljiva, bi lahko postali pomembno metabolno stikalo v proizvodnji rekombinantnih proteinov.

Celice CHO sože vrsto let vodilni ekspresijski sistem za proizvodnjo bioloških zdravil. Trenutni razvoj celičnih linij temelji na naključni integraciji ekspresijskega vektorja v genom celic CHO, s čimer pridobimo klone, ki se med sabo zelo razlikujejo tako v stopnji kot tudi stabilnosti produkcije proteinov. Za razvoj visoko producirajoče celične linije, je tako potrebno generirati in presejati ogromno število klonov. Predpostavili smo, da bi s tarčno integracijo mAb ekspresijskega vektorja v regije genoma celic CHO, ki se nahajajo blizu super-ojačevalcev določenih z metodo ATAC-seq, lažje in hitreje razvili visoko producirajočo celično linijo. V okviru magistrske naloge smo želeli vzpostaviti enostaven in učinkovit način tarčne integracije. Za vstavitev lineariziranega mAb ekspresijskega vektorja v tarčno mesto smo izkoristili popravljalni mehanizem spajanja nehomolognih koncev, ki sodeluje pri popravilu dvojnega preloma DNA. Prelom dvojne vijačnice smo v celicah tarčno sprožili z uporabo orodja za urejanje genoma. Z razvito metodo smo dosegli učinkovitost tarčne integracije med 3 in 11 %, odvisno od učinkovitosti delovanja sistema za urejanje genoma in tarčne regije. V drugem delu magistrske naloge smo pri klonih s tarčno integriranim ekspresijskim vektorjem ovrednotili vpliv super-ojačevalcev na izražanje heterolognih genov ter na produktivnost posameznih klonov. Pokazali smo, da s tarčno integracijo dosežemo 1,7-krat višjo stopnjo izražanja heterolognih genov ter 1,5-krat višjo specifično produktivnost. Z analizo,v kateri smo ugotavljali stopnjo izražanja heterolognega gena na posamezno kopijo ekspresijskega vektorja pa smo ugotovili, da med kloni s tarčno in naključno integracijo ni statistično pomembne razlike. Poleg tega smo pokazali, da z metodo tarčne integracije na osnovi popravljalnega mehanizma spajanja nehomolognih koncev v genom celic CHO integriramo večje število kopij heterolognega gena kot z naključno integracijo. V okviru magistrske naloge smo torej uspeli razviti način za pripravo celičnih linij z izboljšano produktivnostjo. For many years, CHO cells have been the leading expression system for the production of biological drugs. The current cell line development process is based on the random integration of the expression vector into the genome of CHO cells, which leads to the generation of clones that differ significantly in both the rate and stability of protein production. In order to develop a highly producing cell line, it is necessary to generate and screen a huge number of clones. We hypothesized that targeted integration of the mAb expression vector into regions of the genome located close to the super-enhancers, determined by the ATAC-seq method, would allow easier and more rapid development of a high-producing cell line. As part of the master's thesis, we wanted to establish a simple and effective targeted integration approach. For the insertion of a linearized mAb expression vector into the target site, we utilized a DNA double-strand break caused by genome editing tool, which is repaired by the non-homologous end joining(NHEJ). With the developed method, we achieved between 3 and 11 % efficiency of targeted integration, depending on the activity of the genome editing tool and the target region. In the second part of the thesis, we evaluated the influence of super-enhancers on the expression of heterologous genes and the productivity of individual clones with a target-integrated mAb expression vector. Targeted integration has been shown to increasethe expression of heterologous genes and specific productivity of generated clones by 1,7-foldand 1,5-fold, respectively, when compared to random integration. However, when analyzing the mRNA expression levels per copy of the heterologous gene, no statistically significant difference between clones with targeted and random integration was observed. Furthermore, we have shown, for the first time,that when using NHEJ based targeted integration of expression vector into the CHO cell genome, more copies of heterologous genes are introduced into the genome compared to random integration, leading to higher specific productivity. In summary, we developed a targeted integration approach for generating production cell lines with improved productivity.

V zaključni nalogi smo raziskali spreminjanje prostornine celic in pojav napetostnih por celic v različnih hipotoničnih raztopinah vode in gojišča ter v ekvivalentnih hipotoničnih raztopinah saharoze. Pri poskusih smo uporabili epitelne celice kitajskega hrčka [angl. Chinese hamster ovary (CHO) cell], ki so bile gojene v naprednem minimalnem mediju z dodatkom 5% fetalnega telečjega seruma [angl. fetal bovine serum (FBS)] in antibiotikov v CO2 inkubatorju pri 37°C. Vse poskuse smo opravili s konfokalnim mikroskopom, ki omogoča, da naenkrat presvetlimo in zajamemo le določeno rezino celice. Za poskuse spreminjanja prostornine celic smo celicam označili membrano z označevalnim sredstvom CellMask, za opazovanje napetostnih por celic pa smo celice označili s fluorescentnim označevalcem kalcein. Za določanje prostornine celice smo vsebino celotne celice razdelili na skladovnico vodoravnih rezin po osi z, ki so bile med seboj razmaknjene za 0,5 ?m. Spreminjanje prostornine celic smo opazovali v gojišču, v destilirani vodi in v hipotoničnih raztopinah gojišča s 40%, 60%, 80% in 90% deležem vode ter v ekvivalentnih hipotoničnih 23, 65, 125 in 315 mOsm/l raztopinah saharoze. Celice smo posneli pred zamenjavo hipotonične raztopine in po 2, 5, 10, 20, 30, 40, 50 in 60 minutah po dodatku hipotonične raztopine. Prostornino celic smo določili tako, da smo izmerili preseke celic na 7 do 14 enakomerno razmaknjenih rezinah. Celice, pri katerih smo opazovali napetostne pore, smo med poskusom opazovali le v eni izbrani goriščni ravnini. Napetostne pore celic smo opazovali v destilirani vodi in v hipotoničnih raztopinah gojišča s 60%, 80%, 85%, 90% in 95% deležem vode ter v hipotoničnih 23, 65 in 125 mOsm/l raztopinah saharoze. Celice v hipotoničnih raztopinah gojišča z višjimi deleži vode in v destilirani vodi smo slikali vsako drugo sekundo 10 minut po zamenjavi raztopine, celice v hipotoničnih raztopinah z nižjimi deleži vode pa smo slikali vsako trideseto sekundo 1 uro po zamenjavi raztopine. Dobljene rezultate meritev prostornine celic smo pojasnili s teoretičnim modelom, ki je bil razvit na Inštitutu za biofiziko Medicinske fakultete Univerze v Ljubljani. Ugotovili smo, da je nenadno razbarvanje oz. nenaden upad intenzitete celic, označenih s kalceinom, posledica nastanka napetostnih por, kar se na posnetkih jasno vidi. The volume changes and occurrence of tension pores in cells in different hypotonic medium of Leibowitz solution and water and equivalent hypotonic solution of sucrose were observed. Chinese hamster ovary (CHO) cells were used in experiments. The cells were grown in minimal essential medium supplemented with 5% fetal bovine serum (FBS) and antibiotics in CO2 incubator at 37°C. All experiments were performed with confocal microscope, which allows us to illuminate and capture only one specific slice of a cell at once. For cell volume changes we labeled cell membrane with the CellMask marker and for leakage experiments for observing tension pores in cells we labeled cells with the calcein marker. To determine the cell volume, the content of the whole cell was divided into stack of horizontal slices along the z-axis spaced bg 0.5 ?m. The volume changes of the cells were observed in Leibowitz solution, in distilled water, hypotonic media of Leibowitz solution with 40%, 60%, 80%, and 90% water content and equivalent hypotonic solution of sucrose with osmolarities 23, 65, 125 and 315 mOsm/l. The cells were imaged before we replace hypotonic solution and after 2, 5, 10, 20, 30, 40, 50 and 60 minutes after hypotonic solution replacement. The cell volume was determined by determination the area of the cell section at 7 to 14 evenly spaced slices. For leakage experiments the cells were imaged only in one selected plane. The cells tension pores were observed in distilled water, hypotonic media of Leibowitz solution with 60%, 80%, 85%, 90% and 95% water content and equivalent hypotonic solution of sucrose with osmolarities 23, 65 and 125 mOsm/l. The cells in distilled water, in hypotonic media with higher water content and in hypotonic solutions of sucrose with lower osmolarities were imaged every 2 seconds 10 minutes after hypotonic solution replacement. The cells in hypotonic solutions with a lower water content and in hypotonic solutions of sucrose with higher osmolarities were imaged every 30 seconds 1 hour after hypotonic solution replacement. The obtained results of cell volume measurements were explained in terms of a theoretical model, developed on Institute of Biophysics, Faculty of Medicine, University of Ljubljana. We found that sudden decrease of calcein intensity in marker cells due to formation of the tension pores, which can be clearly seen in the recordings.

Rekombinantna tehnologija je omogočila velik napredek na področju bioloških zdravil in zdravljenja mnogih bolezni. Proizvodnja bioloških zdravil poteka v ustreznih ekspresijskih sistemih, med katerimi so najbolj pogosto uporabljene ovarijske celice kitajskega hrčka (CHO), ki so relativno enostavne za genetsko manipulacijo in omogočajo človeku kompatibilne post-translacijske modifikacije proteinov. Trenutne strategije razvoja rekombinantnih celičnih linij temeljijo na naključni integraciji ekspresijskega vektorja v genom. Z razvojem orodij urejanja genoma in vse boljše bioinformatske analize genoma celic CHO pa se razvijajo metode integracije ekspresijskega vektorja v tarčne genomske regije, ki bi potencialno lahko omogočile razvoj visoko producirajočih in stabilnih celičnih linij. V okviru magistrske naloge smo želeli poiskati aktivne regije genoma, kjer se nahajajo regulatorni elementi, ki regulirajo transkripcijo bližnjih genov. Za te regije je značilno, da je kromatin v odprti obliki, saj s tem omogoča dostop transkripcijski mašineriji. Odprte regije kromatina v celicah CHO smo iskali z metodo, ki temelji na uporabi encima modificirane transpozaze, ki dostopne predele kromatina fragmentira in hkrati doda sekvenčne adapterje, ki omogočijo nadaljno analizo DNA fragmentov s sekvenciranjem naslednje generacije. V sklopu optimizacije metode smo določili, da optimalno število celic za pripravo ustrezne knjižnice DNA znaša 5x 104 celic in da moramo za dosego minimalno pomnožene knjižnice zmanjšati število ciklov PCR za dva glede na protokol iz literature. Za analizo sekvenčnih odčitkov smo testirali več bioinformatskih pristopov ter tri različne referenčne genome CHO. Ugotovili smo, da je referenca CHOK1GS_HDv najbolj reprezentativna, saj so se sekvenčni odčitki nanjo prilegali v več kot 94 %. Ponovljivost metode smo potrdili s sekvenciranjem dveh replikatov in močno korelacijo (Pearsonov korelacijski koeficient = 0,95) med regijami, ki so bile skupne obema vzorcema. Po uspešni implementaciji metode na celicah CHO smo odprte regije iskali v treh starševskih celičnih linijah CHO in v dveh časovnih točkah bioprocesa (na dan 3 in dan 7), saj smo želeli določiti univerzalne in stabilno odprte regije. Med odprtimi regijami smo nato identificirali super-ojačevalce in rezultate primerjali z javno dostopno podatkovno bazo transkripcijsko aktivnih regij v celicah CHO. Za več kot polovico identificiranih super-ojačevalcev smo potrdili, da gre res za transkripcijsko aktivne regije. Zaključimo lahko, da je ta metoda določanja odprtih regij kromatina hitra, enostavna in uporabna tudi za identifikacijo super-ojačevalcev. Recombinant technology has facilitated a significant progress in the field of biopharmaceuticals and has opened new doors for treating various diseases. Biopharmaceuticals are produced in different expression systems, where Chinese hamster ovary cells (CHO) are most commonly used, due to their ease of genetic manipulation and human-like post-translation modifications. The current strategies of cell line development for production of recombinant therapeutics are based on a random integration of the transgene into the CHO genome. With the development of genome editing tools and with draft genomes of several CHO cell lines being available, however, targeted integration into genomic hotspots is becoming an option for generating high-producing and stable cell lines. For our master thesis project we wanted to find active regions in the CHO genome, which include regulatory elements and sites of transcription. They are known to be in open sites of chromatin, thus allowing transcription machinery to access the DNA. We determined these accessible regions in CHO cells with the ATAC-seq method that probes and fragments DNA with a modified transposase, which inserts sequencing adapters into open regions of chromatin. During optimization, we determined that the optimal number of cells for obtaining a proper library is 5x 104 and we decreased the number of PCR cycles by 2 cycles compared to the protocol in literature in order to get a better complexity of the library. We tested different bioinformatic algorithms and checked three CHO genome references. The reference CHOK1GS_HDv was the most representative since more than 94 % of our reads aligned to it. We tested the reproducibility of our approach on two replicates and concluded that the method is reproducible since we obtained a similar distribution of fragment length and saw a good correlation (Pearson = 0,95) among regions identified in both replicates. After the successful implementation of the ATAC-seq method on CHO cells we searched for open regions in three CHO parental cell lines at two different time points of the bioprocess (on day 3 and day 7) in order to pinpoint the open regions that are stable and remained open throughout the bioprocess. From those open regions we then identified super-enhancers and compared the results with a publicly available epigenetic study of CHO cells. Among the compared regions, we found that more than half of them really did represent enhancers and concluded that this method is fast, simple, and can be used not only to locate regions of open chromatin, but also to identify super-enhancers.