1,721,005 research outputs found
Evaluacija metoda za pročišćavanje monoklonalnih protutijela proizvedenih iz CHO stanične linije
No full textCHO culture is a well-charaterized cell line that has been used for years in the field of biomanufacturing to produce various valuable pharmaceutical components, such as monoclonal antibodies that are used for the treatment of numerous diseases. Novel, more cost-effective downstream techniques and methods are continuously being developed by large companies. This thesis focused on the characterization of downstream equipment, mainly three components: depth filters (used for filtration), hollow fibers (used for concentration), and ion exchange membrane (used for anionic exchange, or AEX). For the specific process of trastuzumab purification, ErtelAlsop B4E7 filters were proven to be effective in the DNA removal step. In the critical flux experiment, the Microza hollow fibers (0.2 μm pores) could sustain the highest transmembrane flux before significant fouling occurred. Lastly, AEX was effective in removing host cell proteins/DNA.CHO stanice dobro su definirana kultura koje se koriste u proizvodnji mnogih farmaceutskih komponenata, poput monoklonalnih protutijela koja se koriste za prevenciju mnogih bolesti. U svrhu smanjenja troškova, mnoga velika poduzeća kontinuirano razvijaju nove metode pročišćavanja ovih vrijednih spojeva. Tijekom ovog rada ispitana je oprema za pročišćavanje, uz naglasak na tri glavna tipa: dubinski filteri (korišteni za filtraciju), membrane (korištene za koncentraciju) i membranski adsorberi (korištene za anionsku izmjenu). Za specifičan proces pročišćavanja Trastuzumab antitijela, ErtelAlsop B4E7 filteri su dokazano efektivni. Nadalje, ispitivane Microza membrane (pore veličina 0.2 μm) su izdržale najveći protok bez pojave začepljenja. Na kraju, anionska izmjena pokazala se najefektivnijom pri uklanjanju neželjenih staničnih proteina i DNK
Effect of flaxseed cake protein hydrolysates on growth and metabolism of HEK 293 cell line
No full textU posljednje se vrijeme sve veća pažnja pridaje razvoju medija za uzgoj kultura životinjskih stanica koji ne sadrži serum, uslijed uočavanja njegovih brojnih nedostataka. Svoju primjenu u ovim tehnologijama pronašli su proteinski hidrolizati biljnog podrijetla koji su se pokazali kao učinkovit nu-tritivni dodatak. Kao izvor proteina dokazale su se tzv. uljne pogače sjemenki uljarica zaostale nakon postupaka ekstrakcije ulja. Radi se o industrijskom nusproizvodu relativno ograničene iskoristivosti. Cilj je ovog rada bio ispitati učinak proteinskih hidrolizata uljne pogače lana dobivenih pomoću enzima Alcalase, Neutrase i Protamex na rast i metabolizam HEK 293 stanične linije te mogućnost djelomične zamjene seruma u mediju za uzgoj stanica. Rezultati su pokazali da proteinski hidrolizati u koncentraciji od 0,5 g Lˉ¹ i 2,5 g Lˉ¹ mogu poslužiti kao zamjena dijela seruma kada se pripreme cijepanjem enzimom Neutrase. Hidrolizati pripremljeni enzimima Protamex i Alcalase pokazali su manju učinkovitost na rast stanica, pa čak i inhibitorno djelovanje. Manja koncentracija hidrolizata pokazala je bolji učinak na rast stanica nego hidrolizati u većoj koncentraciji.Recently, more and more attention is paid to the development of media for the cultivation of animal cell cultures that do not contain the addition of serum, due to its many disadvantages. Protein hydrolysates of plant origin have found their application in these technologies and have proven to be an effective nutritional supplement. Oil cakes are side-products of oil extraction, an industrial residue rich in protein, have proven to be potential source of proteins to be digested into hydrolysates. The aim of this study was to examine the effect of flaxseed oil cake hydrolysates prepared by enzymes Alcalase, Neutrase and Protamex on the growth and metabolism of HEK 293 cell line, as well as the possibility of partial replacement of serum. The results showed that protein hydrolysates at a concentration of 0,5 g Lˉ¹ and 2,5 g Lˉ¹ can serve as a replacement for part of the serum when prepared by cleavage with the enzyme Neutrase. Hydrolysates prepared by the enzymes Protamex and Alcalase have shown lower efficacy on cell growth and even inhibitory activity. A lower concentration of hydrolysates showed a better effect on cell growth that hydrolysates of higher concentration
Adaptation of CHO-DP12 cells in a nutrient medium supplemented with industrial hemp protein hydrolysates
No full textU današnje vrijeme CHO stanična linija je najatraktivniji i najpopularniji ekspresijski sustav u biotehnološkoj proizvodnji terapeutika. Jedan od najbitnijih koraka optimizacije biotehnološke proizvodnje istih je priprema hranjivog medija za uzgoj. Budući da obogaćivanje medija serumom životinjskog podrijetla nije u skladu sa zahtjevima dobre proizvođačke prakse, kao alternativni dodaci mediju intenzivno se istražuju biljni proteinski hidrolizati. Cilj ovog rada bio je ispitivanje djelovanja proteina izoliranih iz industrijske konoplje na rast i produktivnost IgG producirajuće stanične linije CHODP12. Stoga su hranjivom mediju za uzgoj dodavani enzimski hidrolizati navedenih proteina kao i frakcije hidrolizata (<10 kDa) dobivene pomoću mikrobne proteaze, Neutrase. Ranija istraživanja pokazala su uglavnom nepovoljan utjecaj ovih suplemenata na rast stanica, ali i poboljšanu staničnu produktivnost u odnosu na hidrolizatima netretiranu kulturu CHO-DP12. Zato je također ispitano može li početna prilagodba stanica na navedene suplemente u mediju utjecati na kasniji rast i produktivnost stanične kulture, u smislu boljeg rasta i većeg prinosa IgG.In today's time, the CHO cell line is the most attractive and popular expression system in biotechnological production of therapeutics. One of the most crucial steps in optimizing biotechnological production is the preparation of a nutrient medium for cultivation. Since enriching the medium with animal serum is not in line with the requirements of good manufacturing practice, plant protein hydrolysates are actively being researched as alternative additives to the medium. The aim of this study was to investigate the effect of proteins isolated from industrial hemp on the growth and productivity of the IgG-producing cell line CHO-DP12. Therefore, enzymatic hydrolysates of the mentioned proteins as well as hydrolyzate fractions (<10 kDa) obtained using the microbial protease, Neutrase, were added to the nutrient culture medium. Earlier studies showed a mostly unfavorable influence of these supplements on cell growth, but also improved cell productivity compared to the CHO-DP12 culture not treated with hydrolysates. Therefore, it was also examined whether the initial adaptation of the cells to the mentioned supplements in the medium can affect the subsequent growth and productivity of the cell culture, in terms of better growth and higher IgG yield
Preparation of natural eutectic solvents and their application in cryoprotection of cell lines
Get PDFPrirodna eutektična otapala (eng. Natural Deep Eutectic Solvents, NADES) su nova generacija ekološki prihvatljivih otapala sa mogućim primjenama u različitim industrijskim područjima. Definiraju se kao smjesa dviju ili više komponenata koje se međusobno mogu povezati vodikovim vezama te koje u određenom omjeru imaju niže talište nego pojedinačne komponente smjese. S obzirom na njihovu široku primjenu, u ovom radu ispitana je mogućnost njihove primjene kao krioprotektanata na staničnoj liniji HEK 293T. U ovom radu pripravljena su dva različita NADES-a u različitim molarnim odnosima (kolin klorid:glukoza, prolin:glukoza) te s različitim udjelima vode. Učinkovitost ovih NADES-a kao krioprotektanata uspoređena je sa standardnim krioprotektantima, glicerolom i dimetil sulfoksidom. Istraživanje je pokazalo da testirani spojevi nisu dostojna zamjena standardnim kriprotektantima.Natural deep eutectic solvents (NADES) are new generation of environmentally friendly solvents with potential application in various industrial fields. They are defined as a mixture of two or more components connected to each other by hydrogen bonds. At a certain ratio of their building components, NADES can have lower melting points than the individual components of the mixture. Considering their widespread application, NADES are tested as cryoprotectants on cell line HEK 293T. Two different NADES are made for this experiment; they are consisted of proline/glucose and choline chloride/glucose in different molar ratio and with different percentage of water content. Efficiency of those NADES are compared with standard cryoprotectants, glycerol and dimethyl sulfoxide. The results showed that NADES cannot serve as an appropriate alternative to the standard cryoprotectants
The production of recombinant proteins in suspensive CHO cell line
Get PDFCHO stanična linija pogodna je za korištenje u tehnologiji stanica jer se može genetički modificirati i može proizvoditi rekombinantne proteine u svrhu istraživanja te se uzgajati u suspenziji. Cilj ovog rada je bio ispitati učinak različitih sastava medija na rast stanične linije, utrošak glukoze te proizvodnja proteina. U ovom radu ispitivan je rast suspenzijske CHO stanične linije u mediju s 5 % seruma te 1% seruma te 4% sojinog hidrolizat. Najveću specifičnu brzinu rasta pokazao je medij s 1% seruma i 4% sojinog hidrolizata te je u tom uzorku zabilježena najveća potrošnja glukoze od 17,372 mM.CHO cell line is suitable for use in cell culture technology due to its genetic modification that made them immortalized and suitable for production of recombinant proteins for research and therapeutic purposes. The cell line grow as either adherent or suspension culture. The aim of this research was to examine the effect of different medium component on the growth of cell lines, the consumption of glucose and on the protein production. In this research was examined growth of suspension of CHO cell line in medium containing 5% of serum and second suspension containing 4% of soy hydrolyzate and 1% of serum respectively. The maximum specific growth speed occured in medium containing 1% of serum and 4% of soy hydrolyzate but the same medium also showed highest glucose consumption rate of 17,372 mM
Effect of flaxseed cake protein hydrolysate on growth and productivity of CHO DP-12 cells
No full textPosljednjih desetljeća uočeni su rizici vezani uz korištenje seruma u proizvodnji terapeutika pomoću kultura životinjskih stanica, pa se sve više nastoje razviti mediji bez dodatka seruma. Hidrolizati osiguravaju važne nutrijente stanicama, a osim što mogu potaknuti rast staničnih linija, istraživanja su pokazala da pozitivno utječu i na produktivnost staničnih linija. Cilj ovog rada bio je ispitati učinak proteinskih hidrolizata, dobivenih pomoću enzima Alcalase, Neutrase i Protamex, uljne pogače lana na rast i produktivnost stanične linije CHO DP-12, proizvođača humanog protutijela imunoglobulina (IgG) anti IL-8. Također, istražena je i mogućnost zamjene dijela seruma proizvedenim hidrolizatima. Istraživanje je pokazalo da sva tri proteinska hidrolizata pozitivno djeluju na rast stanica, no samo proteinski hidrolizat dobiven pomoću Alcalase ima statistički značajan pozitivan učinak na produktivnost CHO DP-12 stanične linije. Hidrolizati lana mogu se koristiti kao djelomična zamjena za serum s obzirom da je dodatkom i najmanjeg volumnog udjela hidrolizata postignut dobar stanični rast.Scientists are trying to develop a serum free medium because of the risks associated with the use of serum in the production od therapeutics using animal cell cultures. Hydrolysates provide important nutrients to the cells and cause growth stimulation and positive effect on the productivity of cell lines. The aim of this study was to investigate the effect of protein hydrolysates from flaxseed cake obtained by the Alcalase, Neutrase and Protamex enzymes on the growth and productivity of CHO DP-12 cell lines, producers of immunoglobulin (IgG) anti IL-8. Also, the possibility of replacing part of serum with hydrolysates was investigated. Research has shown that all protein hydrolysates positively affect cell growth but only the protein hydrolysate obtained with Alcalase has a statistically significant positive effect on the productivity. Flax hydrolysates can be used as a partial substitution for serum since a good cell growth has been achieved by addition of the smallest volume of hydrolysates
Effect of protein isolates from flaxseed meal on animal cell culture growth and productivity
Get PDFMnoge industrije sve više teže za efikasnijim iskorištavanjem organskih ostataka nastalih nakon prerade proizvoda iz različitih grana poljoprivrede i proizvodnje hrane. Kao jedan od takvih organskih ostataka s velikim potencijalom primjene ističe se uljna pogača koja zaostaje nakon ekstrakcije ulja. Pogače lana, zbog svog sastava proteina, predstavljaju potencijalnu sirovinu za primjenu u tehnologiji životinjskih stanica. Stoga je u ovom radu ispitan utjecaj proteinskih izolata iz uljne pogače lana na rast i produktivnost CHO-RP stanične linije koja proizvodi rekombinantni protein. Također, ispitan je utjecaj proteinskih izolata i na rast HEK-293T stanične linije. Budući da serum, kao standardni nutritivni suplement u hranjivom mediju, ima određenih nedostataka, istražena je mogućnost korištenja proteinskih izolata kao potencijalna zamjena za serum. Istraživanje je pokazalo da dodatak proteinskih izolata u koncentracijama od 0,1 g L-1 i 0,2 g L-1 u hranjivi medij ima pozitivan učinak na rast i produktivnost CHO-RP stanica, dok u koncentracijama većim od 1 g L-1 dolazi do inhibicijskog djelovanja na rast obje stanične linije. Također je dokazano da se proteinski izolati uljne pogače lana ne mogu koristiti kao djelomična zamjena za serum jer nisu pokazali utjecaj na rast stanica u mediju s 1% seruma.A lot of industries are looking for efficient exploitation of organic residues from various domain of agriculture and food manufacturing. Oil meal, a substance that remains after oil extraction from seeds, is one of these organic residues with great potential of application. Because of its protein content, the flaxseed meal can potentionally be used in animal cell technology. Therefore, in this work, the influence of protein isolates from flaxseed meal on growth and productivity of a recombinant protein producing cell line (CHO-RP) was investigated. The effect of protein isolates on growth of HEK-293T cell line was observed as well. Since the serum, as a standard nutritional supplement in culture media, has certain disadvantages, protein isolates were investigated as possible serum replacement. The results of the research have shown that the addition of protein isolates at concentrations of 0.1 g L-1 and 0.2 g L-1 in cell culture medium has positive effect on growth and productivity of CHO-RP cells. However, when concentration of protein isolates in medium is above 1 g L-1 there is an inhibitory effect on cell growth. Also, it has been proven that protein isolates from flaxseed meals cannot be used as a partial substitution for serum because they have not shown the effect on cell growth in 1% serum media
Evaluacija metoda za pročišćavanje monoklonalnih protutijela proizvedenih iz CHO stanične linije
No full textCHO culture is a well-charaterized cell line that has been used for years in the field of biomanufacturing to produce various valuable pharmaceutical components, such as monoclonal antibodies that are used for the treatment of numerous diseases. Novel, more cost-effective downstream techniques and methods are continuously being developed by large companies. This thesis focused on the characterization of downstream equipment, mainly three components: depth filters (used for filtration), hollow fibers (used for concentration), and ion exchange membrane (used for anionic exchange, or AEX). For the specific process of trastuzumab purification, ErtelAlsop B4E7 filters were proven to be effective in the DNA removal step. In the critical flux experiment, the Microza hollow fibers (0.2 μm pores) could sustain the highest transmembrane flux before significant fouling occurred. Lastly, AEX was effective in removing host cell proteins/DNA.CHO stanice dobro su definirana kultura koje se koriste u proizvodnji mnogih farmaceutskih komponenata, poput monoklonalnih protutijela koja se koriste za prevenciju mnogih bolesti. U svrhu smanjenja troškova, mnoga velika poduzeća kontinuirano razvijaju nove metode pročišćavanja ovih vrijednih spojeva. Tijekom ovog rada ispitana je oprema za pročišćavanje, uz naglasak na tri glavna tipa: dubinski filteri (korišteni za filtraciju), membrane (korištene za koncentraciju) i membranski adsorberi (korištene za anionsku izmjenu). Za specifičan proces pročišćavanja Trastuzumab antitijela, ErtelAlsop B4E7 filteri su dokazano efektivni. Nadalje, ispitivane Microza membrane (pore veličina 0.2 μm) su izdržale najveći protok bez pojave začepljenja. Na kraju, anionska izmjena pokazala se najefektivnijom pri uklanjanju neželjenih staničnih proteina i DNK
CHO cell growth in basal nutrient medium with flaxseed protein hydrolysate and albumin
No full textU ovom radu obrađena je tema uzgoja adherentnih stanica ovarija kineskog hrčka CHO DP- 12 u bazalnom hranjivom mediju (DMEM) sa sljedećim dodacima: 10% fetalnog goveđeg seruma (standard), s dodatkom albumina i hidrolizata proteina uljne pogače lana te s dodatkom samog albumina. Albumin je glavni protein u fetalnom goveđem serumu te se ispituje njegov značaj za rast i održavanje stanica, dok se biljni proteinski hidrolizati često dodaju kao zamjena proteinima životinjskog podrijetla. Krajnji cilj ovakvih ispitivanja jest pronaći optimalan medij za uzgoj stanica sa što manjom količinom dodanog seruma, budući da postoji mnogo problema vezanih za upotrebu seruma kao što su cijena, sigurnost, potencijalne kontaminacije te etička pitanja vezana za proizvodnju seruma. Eksperiment je proveden na način da su stanice uzgajane u navedenim medijima te je određenih dana
bojilom Trypan plavo praćen rast stanica. Rezultati pokazuju da je najveća koncentracija i najbrži rast u mediju koji sadrži 10% fetalnog goveđeg seruma, a najmanja koncentracija u mediju koji je sadržavao samo albumin. Albumin sa ili bez dodanih lanenih hidrolizata nije jednakovrijedna zamjena za fetalni serum.The subject of this thesis is culturing adherent chinese hamster ovary cell line CHO DP-12 in basal culture media (DMEM) containing different supplements: 10% fetal bovine serum (standard), with different concentrations of albumin and protein hydrolysates and with the addition of albumin itself. Albumin is the major protein in fetal bovine serum and its effect on cell growth and maintenance is examined in order to find the optimal medium for culturing serum-free cells, as there are many problems regarding serum use such as price, safety, potential contamination, and ethical issues related to serum production. The experiment was carried out in such a way that the cells were grown in the previously mentioned media, and on certain days the cells were counted using the Trypan blue method, thus monitoring their growth and development. The results show that the highest concentration and the fastest growth is in the medium containing 10% of fetal bovine serum, and the lowest concentration in the medium containing only albumin. Supplementation of DMEM with albumin and/or flaxseed hydrolysates cannot is not recommended as serum replacement
Biocompatibility study of extracellular liver matrix and electrospun poli-ε-caprolactone scaffolds
No full textRazvojem tehnika tkivnog inženjerstva omogućila se izrada nosača poželjnih mehaničkih i bioaktivnih svojstava potrebnih za regeneraciju tkiva. Dok alternativu predstavljaju tehnike transplantacijske medicine, metode tkivnog inženjerstva prednjače u in vitro modelima za istraživanje raka te razvoja farmaceutika. Cilj ovog rada bio je ispitati i usporediti biokompatibilnost elektroispredanog sintetskog i kompozitnog nosača sa prirodnim nosačem, nacijepljenih s HeLa stanicama. Nosači su bili izrađeni od polikaprolaktona (PCL), PCL-a kombiniranog sa svilinim fibroinom (PCL-SF) te izvanstaničnog matriksa svinjske jetre (ECM). U svrhu istraživanja, testovi biokompatibilnosti nosača sastojali su se od analize uspješnosti nacjepljivanja stanica te dinamike rasta i transfekcije. Kroz sva tri testa, najbolje rezultate je dao nosač od PCL-SF, dok su najmanje uspješni bili oni na ECM nosaču. Rezultati s primjenom kulture HeLa stanica u izradi modelnog tkiva pokazuju da je tehnologija nano-vlakana napredna tehnika u stvaranju staničnih nosača koji uspješno oponašaju potporanj prirodnih tkiva.The development of tissue engineering enabled the production of cell scaffolds with superior mechanical and bioactive characteristics needed for tissue regeneration. While regenerative medicine offers an acceptable alternative, current methods in tissue engineering still lead the way in in vitro cancer model research as well as drug development. The aim of this study was to test and compare biocompatibility of HeLa seeded, electrospun polymeric synthetic and composite scaffolds with a natural scaffold. The scaffolds were made of polycaprolacton (PCL), PCL composite integrated with silk fibroin protein (PCL-SF) and extracellular matrix isolated from pork liver (ECM). In studying the biocompatibility of scaffolds, three tests were performed: cell seeding efficiency, growth dynamics and transfection efficiency. PCL-SF proved the most successful in all three tests, while porcine ECM performed the worst. HeLa culture results indicate the nano-fibre technology as an advanced technique that is indeed able to successfully mimic the 3D architecture of native tissues
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