Monitoring and analysis of dynamic growth of human embryonic stem cells : comparison of automated instrumentation and conventional culturing methods

Abstract Background Human embryonic stem cells (hESCs) are a potential source of cells for use in regenerative medicine. Automation of culturing, monitoring and analysis is crucial for fast and reliable optimization of hESC culturing methods. Continuous monitoring of living cell cultures can reveal more information and is faster than using laborious traditional methods such as microscopic evaluation, immunohistochemistry and flow cytometry. Methods We analyzed the growth dynamics of two hESC lines HS237 and HS293 in a conventional culture medium containing serum replacement and a xeno-free X-vivo 10 medium. We used a new automated culture platform utilizing machine vision technology, which enables automatic observation, recording and analysis of intact living cells. We validated the results using flow cytometry for cell counting and characterization. Results In our analyses, hESC colony growth could be continuously monitored and the proportion of undifferentiated cells automatically analyzed. No labeling was needed and we could, for the first time, perform detailed follow up of live, undisturbed cell colonies, and record all the events in the culture. The growth rate of the hESCs cultured in X-vivo 10 medium was significantly lower and a larger proportion of the cells were differentiated. Conclusion The new automated system enables rapid and reliable analysis of undifferentiated growth dynamics of hESCs. We demonstrate the effectiveness of the system by comparing hESC growth in different culture conditions

A Defined and Xeno-Free Culture Method Enabling the Establishment of Clinical-Grade Human Embryonic, Induced Pluripotent and Adipose Stem Cells

Peer reviewe

Efflux Protein Expression in Human Stem Cell-Derived Retinal Pigment Epithelial Cells

Retinal pigment epithelial (RPE) cells in the back of the eye nourish photoreceptor cells and form a selective barrier that influences drug transport from the blood to the photoreceptor cells. At the molecular level, ATP-dependent efflux transporters have a major role in drug delivery in human RPE. In this study, we assessed the relative expression of several ATP-dependent efflux transporter genes (MRP1, -2, -3, -4, -5, -6, p-gp, and BCRP), the protein expression and localization of MRP1, MRP4, and MRP5, and the functionality of MRP1 efflux pumps at different maturation stages of undifferentiated human embryonic stem cells (hESC) and RPE derived from the hESC (hESC-RPE). Our findings revealed that the gene expression of ATP-dependent efflux transporters MRP1, -3, -4, -5, and p-gp fluctuated during hESC-RPE maturation from undifferentiated hESC to fusiform, epithelioid, and finally to cobblestone hESC-RPE. Epithelioid hESC-RPE had the highest expression of MRP1, -3, -4, and P-gp, whereas the most mature cobblestone hESC-RPE had the highest expression of MRP5 and MRP6. These findings indicate that a similar efflux protein profile is shared between hESC-RPE and the human RPE cell line, ARPE-19, and suggest that hESC-RPE cells are suitable in vitro RPE models for drug transport studies. Embryonic stem cell model might provide a novel tool to study retinal cell differentiation, mechanisms of RPE -derived diseases, drug testing and targeted drug therapy

Screening ethnically diverse human embryonic stem cells identifies a chromosome 20 minimal amplicon conferring growth advantage

The International Stem Cell Initiative analyzed 125 human embryonic stem (ES) cell lines and 11 induced pluripotent stem (iPS) cell lines, from 38 laboratories worldwide, for genetic changes occurring during culture. Most lines were analyzed at an early and late passage. Single-nucleotide polymorphism (SNP) analysis revealed that they included representatives of most major ethnic groups. Most lines remained karyotypically normal, but there was a progressive tendency to acquire changes on prolonged culture, commonly affecting chromosomes 1, 12, 17 and 20. DNA methylation patterns changed haphazardly with no link to time in culture. Structural variants, determined from the SNP arrays, also appeared sporadically. No common variants related to culture were observed on chromosomes 1, 12 and 17, but a minimal amplicon in chromosome 20q11.21, including three genes expressed in human ES cells, ID1, BCL2L1 and HM13, occurred in >20% of the lines. Of these genes, BCL2L1 is a strong candidate for driving culture adaptation of ES cells

The Optimization of GMP/GLP Laboratories: : The laboratory utilization and work safety improvement project

The pharmaceutical industry is regulated by the Good Manufacturing Practices (GMP) and Good Laboratory Practices (GLP) in order to minimize the risks that might have an impact on the safety of the patients. The purpose of the regulations is to assure, that the pharmaceutical products meet the safety requirements and have the intended product identity, quality and purity characteristics. Regulations are regulating and covering the whole manufacturing process from the used premises and starting materials to the manufacturing process and final product and disposal of the product. This study aim was to optimize the pharmaceutical biotechnology company FIT Biotech’s Finn-Medi 3 building laboratory premises to meet the assessed quality requirements. The study objectives was to evaluate how to implement the appropriate quality requirements of different laboratory activities in accordance with GLP/GMP regulations. Additionally laboratory system efficiency, working practices and work safety was evaluated before and after the optimization of the laboratories was performed. The impact of the laboratory optimization process was studied through internal audits and the questionnaire was used as a self-evaluation tool for evaluating personnel perspectives of the optimization project. The experimental work consisted of the laboratory optimization project, where selected laboratory rooms were optimized to a higher quality level as deemed necessary by their intended use. As a result of the optimization project, all optimized laboratory rooms were meeting the set quality level at the time of the second audit. Only few quality related documents were still under updating at the time of the performance of the second audit. The quality level of the Finn-Medi 3 laboratory premises were noticed to be improved and conducting the audit. Audit as a method of evaluation, was noticed to be sufficient for detecting even small deficiencies in the laboratories. Questionnaire results partly supported the audit results, but due to the limited participants wich completed the queries it was difficult to draw any conclusions from the feedback obtained. However, from the evaluation of the completed questionnaires it was indicative that the optimization project influenced to the improved quality level of premises and work practices and result verified also by the audit conducted.Lääketeollisuudessa lääkkeiden valmistus on tarkoin säädeltyä GMP (Good Manufacturing Practice) ja GLP (Good Laboratory Practice) säädöksissä. Sääntelyn tarkoituksena on vähentää lääkkeiden käytöstä johtuvaa turvallisuusriskiä ja varmistaa, että lääkinnällinen tuote vastaa asetettuja turvallisuusmääräyksiä ja tuotteella on sille tarkoitetut ominaisuudet sekä että se täyttää sille asetetut laatu- ja puhtausvaatimukset. Säädökset kattavat koko tuotantoprosessin tuotantotiloista ja raaka-aineista aina valmiiseen tuotteeseen ja tuotteen hävitykseen saakka. Tässä työssä bioteknologia lääkeyrityksen FIT Biotech:n Finn-Medi 3 rakennuksessa sijaitsevat laboratoriotilat optimoitiin vastaamaan niille asetettuja tiukentuneita laatuvaatimuksia. Työn tarkoituksena oli kartoittaa kuinka päivitetyissä laboratoriotiloissa GLP/GMP laatuvaatimukset voitaisiin parhaiten toteuttaa. Laboratorioiden toiminta, työkäytännöt ja työsuojeluun liittyvät asiat kartoitettiin ennen ja jälkeen laboratorioiden optimoinnin. Henkilökunnan kokemuksia laboratorioiden optimointiprojektista kartoitettiin laboratoriotilojen päivityksen jälkeen tehtävällä kyselyllä. Työn käytännön osuuden muodosti laboratorioiden optimointi projekti, jossa tietyt laboratoriohuoneet optimoitiin vastaamaan nykyistä tarkoitustaan. Optimoinnin jälkeisessä auditoinnissa todettiin, että kaikki muutetut laboratoriohuoneet täyttivät niille asetetut laatuvaatimukset. Kokonaisuudessaan Finn-Medi 3:n laboratoriotilojen laadun todettiin parantuneen ja auditoinnin todettiin olevan sovelias menetelmä GLP/GMP sääntöjen noudattamisen kartoittamiseksi. Auditoinnissa ilmeni vain muutamia laadunvalvontaan liittyvien dokumenttien puuttumista. Kyselyn tulokset tukivat osittain auditoinnista saatuja tuloksia, mutta kyselyn perusteella ei voitu tehdä lopullisia johtopäätöksiä optimoinnin vaikutuksesta kyselyyn vastanneiden vähäisen henkilömäärän vuoksi. Kyselyn tulokset kuitenkin viittasivat siihen, että laboratorioiden optimoinnilla on ollut positiivisia vaikutuksia laadun parantumisen ja työkäytäntöjen kehittymisen suhteen