File Name: introduction to cell culture theory and technique .zip
Essentially, cell culture involves the distribution of cells in an artificial environment in vitro which is composed of the necessary nutrients, ideal temperature, gases, pH and humidity to allow the cells to grow and proliferate. Whereas pieces of tissue can be put in the appropriate culture to produce cells that can then be used for culture explant culture , cells from tissues soft tissue can be obtained through enzymatic reactions. Such enzymes as trypsin and proname are used to break down the tissue and release the desired cells.
However, researchers are struggling to improve the scalability, reproducibility and quality of this descriptive disease modelling. Addressing these limitations will be the first step towards a new era in hiPSC research — that of predictive disease modelling — involving the correlation and integration of in vitro experimental data with longitudinal clinical data.
This approach is a key element of the emerging precision medicine paradigm, in which hiPSCs could become a powerful diagnostic and prognostic tool.
Here, we consider the steps necessary to achieve predictive modelling of neurodegenerative disease with hiPSCs, using Huntington disease as an example. Since refs 1 , 2 , the validation of cellular reprogramming in human cells has opened the gates for a wealth of applications in stem cell biology, disease modelling, drug discovery and regenerative medicine 3.
The ability to generate virtually any cell type by differentiating in vitro hiPSCs is particularly relevant in the field of neuroscience, owing to the limited access to primary cells from the human CNS and peripheral nervous system. Disease modelling platforms based on hiPSCs have also been used for drug repositioning, a practice that builds on previous toxicological and safety studies to find new applications for known drugs For example, disease modelling experiments on hiPSCs derived from individuals with amyotrophic lateral sclerosis ALS were used to identify the anti-epileptic drug ezogabine as a potential treatment for ALS 13 , The advantage of hiPSCs for this kind of treatment is that they can be generated from the same individual who will receive the therapy, thus minimizing the problem of graft rejection In contrast, many more studies aim to use patient-derived iPSCs for disease modelling as listed on ClinicalTrials.
This Perspective addresses the need for a new approach to the use of hiPSC lines, particularly in in vitro disease modelling.
Indeed, two specific lines H9 and H1 accounted for Notably, derivation methods, the quality and type of the starting material, culture conditions and differentiation protocols are not standardized across laboratories, thus complicating the interpretation of hiPSC-derived modelling data. This inconsistency was also highlighted by an analysis of the literature describing the in vitro generation of dopaminergic neurons, the neuronal subtype most frequently derived from human pluripotent stem cells both hESCs and hiPSCs Almost half of these publications described new differentiation protocols or substantial modifications to pre-existing protocols, resulting in a total of 74 different methods for generating human dopaminergic neurons in vitro.
However, only five of these 74 methods were substantially re-used by other research groups; the two most-cited publications were those published in journals with the highest impact factors 21 , 22 , Although this process is a natural part of the scientific inefficiency of a nascent field, we should be aware that it could also disperse effort and resources.
Clearly, improving existing differentiation protocols and developing alternative approaches is extremely valuable. However, we should also direct resources towards the identification and use of robust indicators of the desired cell type, including genetic and proteic markers, and electrophysiological characteristics. This approach would enable us to funnel research efforts towards the achievement of high-quality products, increasing the efficiency of the field.
Several other factors contribute to the complexity involved in using hiPSCs for disease modelling. These alterations can have profound consequences; for example, the retention of DNA methylation signatures characteristic of the parent cells — known as somatic memory — can restrict the differentiation potential of hiPSCs 26 , Second, reprogramming to pluripotency almost completely erases the epigenetic age of the donor 28 , 29 , 30 , although some epigenetic and mitochondrial signatures carried by cells from elderly donors can still be found in hiPSCs 28 , This removal of the majority of epigenetic landmarks renders hiPSCs similar to hESCs, and thus suitable for modelling developmental mechanisms and disorders, but poses some difficulties when attempting to recreate the status of an aged cell.
Last, the variability encountered in the derivation of hiPSCs from human tissue is further increased by the variability introduced by the subsequent in vitro differentiation protocols, which can differ greatly between laboratories. We believe that the stem cell research community has the skills to tackle these different sources of variability and the resulting loss of time, human resources and funds, and to leverage the full potential of hiPSC disease modelling.
However, reliable descriptive and predictive disease modelling can be achieved only through the establishment of a high-quality protocol along clear guidelines, enabling a higher degree of data sharing and collaboration between laboratories, clinicians and industries. Given the high degree of variability involved in the establishment of hiPSC lines, the standardized, methodical characterization of the starting cell population and resulting hiPSC lines, along with a high degree of protocol transparency, will be essential for developing new, shareable disease modelling tools.
Ideally, for any given target disease, we should strive towards a standardization of the conditions for derivation, culturing, storing and differentiation of hiPSC lines. A practical example of the relevance of accurate phenotyping comes from the Parkinson disease PD research field, where specific cellular markers were found to correlate with the transplantation efficiency of dopaminergic progenitors in rats.
The identification and use of these markers led to improvements in transplantation outcome and reproducibility 32 , Prestigious scientific societies and international consortia had, and continue to have, an important role in promoting the excellence of stem cell science and its applications.
For example, since the International Society for Stem Cell Research has issued hESC research guidelines with the aim of optimizing the use of these cells in preclinical and clinical studies 34 , Additionally, the New York Stem Cell Foundation repository contains a collection of disease-specific stem cell lines, some accompanied by a full genomic sequence, and the California Institute for Regenerative Medicine is in the process of collecting a large number of healthy and diseased tissues for the generation of an hiPSC repository with de-identified clinical and demographic information see Related links.
These entities all responded to the need for a unified framework to enable the sharing of high-quality cells across multiple stakeholders. These details are often omitted from conventional research articles.
We propose that the field should take the approaches described above as examples to generate one worldwide hiPSC certification system to confer the equivalent of a warranty label on each newly generated hiPSC line. This system would work in a similar way to the ISO quality management systems This process would be similar to that employed by J. Most importantly, this process would create a unified and global real-time database that, for any given cell line, synthesizes research data from a range of sources.
In addition to the creation of an hiPSC certificate, the performance of hiPSC lines in standard directed differentiation protocols should be validated. Therefore, we should define experimental end points that represent the benchmarks that a differentiating hiPSC must achieve to qualify as a differentiated neuron, hepatocyte or cardiomyocyte. Similarly, the coalescence of specific directed differentiation benchmarks into unified experimental end points would allow the establishment of certified and recognized cell lines and protocols, which would greatly advance the knowledge on differentiated patient-derived hiPSCs as they would stem from a common high-quality and state-of-the-art pipeline.
In our opinion, the adoption of these ambitious models is the gateway to predictive disease modelling. Indeed, making a reliable prediction requires a stable and defined starting assumption, which is why we need to increase the level of information we have on the hiPSC lines currently in use.
After the discovery of reprogramming technology, research into neurological disorders 42 , 43 , 44 , 45 and neurodegenerative diseases 46 , 47 , 48 , 49 initially focused on establishing whether the reprogramming process was equally efficient in cells from healthy individuals and in cells from individuals with disease.
The aim of this work was to ensure that hiPSCs would provide a valid system for modelling disease. During this phase of research, a profound transformation in the technical aspects of the procedure occurred, the most relevant aspect of which was the transition from integrative to non-integrative delivery systems 3. Thereafter, a progressive interest in leveraging hiPSC technology as a disease modelling platform was accompanied by an increase in the number of studies that used multiple hiPSC lines, and more precise and robust differentiation strategies, to minimize the effect of their intrinsic variability 42 , 43 , 44 , 45 , 46 , 47 , 48 , Unfortunately, barriers to the use of hiPSC-based disease modelling for more than the straightforward comparison of control and disease-perturbed regulatory networks remain.
First, the patient-derived hiPSCs available to preclinical researchers generally do not represent the whole spectrum of manifestations for a given disease, which can bias study results. Instead, sampling should take into account the diverse presentations that a disease can have; for example, the clinical manifestations of idiopathic diseases such as PD are influenced by the genetic background of the individual as well as environmental factors Similarly, individuals with monogenic diseases such as HD can exhibit different combinations of a wide range of symptoms, including motor and psychiatric disorders Indeed, in our experience, the majority of publications on patient-derived hiPSCs — including our own — include extremely limited information on the donor patient.
This lack of information prevents researchers from being able to stratify hiPSC lines according to patient characteristics, confuses the interpretation of the resulting in vitro data and frustrates attempts at conducting reliable meta-analyses.
As the number of donor patients is generally limited, in vitro hiPSC differentiation studies seem to often be underpowered, and correcting cells with gene editing to achieve a control cell line with the same genetic background as the patient cell line is an inferior substitute for a population of patient and control cells large enough for an adequately controlled and well-powered study.
If we attempt a comparison with clinical trials, we find that the interventional studies for AD listed as currently active on ClinicalTrials. The appropriate cohort size depends on the aims of the specific study and intervention; however, until hiPSC studies of familial and sporadic AD used four or fewer distinct hiPSC lines or clones Unfortunately, this limitation is not unique to research into idiopathic conditions such as AD but is also observed in hiPSC studies of genetic diseases for example, HD 52 and complex psychiatric conditions for example, schizophrenia 45 , both of which are usually studied in clinical trials that include large cohorts of participants who are carefully stratified to maximize signal-to-noise ratio.
In direct contrast to clinical trials, sample size estimation and power analyses are not required when planning in vitro experiments, even if those experiments use patient-derived cells. In our opinion, power analysis should be a mandatory element of the planning of hiPSC studies. In clinical trials and animal studies, ethical considerations are the main reasoning behind mandatory power analyses — it is unethical to perform an inadequately powered study.
We believe that the costs involved in the derivation, maintenance, differentiation and analysis of hiPSCs are important limiting factors that should be viewed in a similar way to these ethical considerations. Nevertheless, a priori sample sizing for cell-based in vitro studies is challenging because it needs to account for many variables, including the nature of the disease multifactorial versus monofactorial , the differentiation protocol and the variability of the readouts, which make the resulting estimation highly unreliable.
Unfortunately, in our experience the results of this computation often greatly exceed the number of available cell lines or impose a substantial economic burden to the experiment. Nevertheless, it would be highly desirable to identify and implement power analysis methodologies that can aid the design of in vitro experiments. The feasibility of performing hiPSC experiments with large sample sizes is substantially influenced by the available cell-handling approaches and experimental throughput.
For example, the need for hiPSC-based studies with sample sizes large enough to represent the diversity of the patient population discourages the use of traditional low-content approaches.
Technological advances in automated cell manipulation 54 , 55 , microfluidic systems 56 , 3D bioprinting 57 , 58 , organ-on-chip 59 and organoids 60 , 61 can boost experimental throughput to hundreds of lines or clones while maintaining readouts with single-cell resolution. To reach this goal, we need closer collaboration between clinicians and preclinical researchers to enable the definition of appropriate sample sizes, keeping in consideration the epidemiological characteristics of the disease of interest.
Moreover, a change in policies governing the use of donor patient clinical records is highly desirable 62 ; better access to these records for preclinical researchers would enable us to gain more information from descriptive modelling studies and move us towards predictive modelling.
Modelling diseases in vitro is key to uncovering prognostic and predictive biomarkers at relevant surrogate end points. This knowledge is important for the development of preventive treatment approaches. To this end, there is a strong need to establish the most translatable and predictive in vitro cellular models, which we believe can be achieved via the precision medicine model.
Precision medicine is a relatively new operative paradigm that strives to optimize disease prevention and therapy by taking a predictive and preventive approach, as opposed to the reactive approach that is the current standard. The final goal of precision medicine is to predict the individual disease trajectory of each patient and to precisely intervene when the disease processes are preventable or reversible We believe that predictive disease modelling with hiPSCs should be considered in the context of the precision medicine guidelines 64 , 65 , and should encompass longitudinal studies that involve the stratification of patients and their cells, and the use of computational models to integrate data from different time points and generate information on disease trajectory.
Stratification is a statistical procedure that splits a mass into several layers by grouping together units with common characteristics, and is a key aspect of precision medicine. The stratification of patients on the basis of a detailed molecular assessment, including biomarker analyses as well as the collection of genetic, epigenetic, phenotypic and psychosocial data 66 , enables the heterogeneity of complex multifactorial diseases to be broken down into simpler elements.
Similar approaches are beginning to be applied in neurology; for example, genomic data has been successfully used to stratify individuals with familial ALS 72 with the aim of delivering different therapies to different patient cohorts.
Today, the intrinsic variability of hiPSCs and the scarcity of hiPSC lines derived from single patients or patient cohorts compels us to average data from several lines to increase the strength of the recorded biological data. However, this approach might prevent the identification of biological mechanisms that are specific to a single donor or patient cohort.
Therefore, during the creation of disease-specific hiPSC libraries, accurate molecular stratification of patients should be performed to ensure that hiPSC lines are generated from clinically relevant, homogeneous cohorts of patients. In the other direction, omics-based fingerprinting of patient hiPSCs could inform clinical-level patient stratification. Longitudinal follow-up of patients is an essential aspect of both the development and the implementation of precision medicine.
Follow-up enables the disease trajectory of a cohort of patients to be studied with the aim of predicting future decline and intervening before the development of full symptomatology. For example, the Alzheimer Precision Medicine Initiative has established several experimental cohorts that include participants at a range of disease stages, from early asymptomatic individuals to patients with late-stage AD In parallel with longitudinal patient follow-up, a cohort of patient-derived hiPSCs can be followed in vitro on a much shorter timescale and with the opportunity to modify the cell environment.
This approach unlocks the full potential of longitudinal studies by enabling the correlation of in vivo and in vitro readouts. Therefore, sharing patient-level data is vital not only for the correct interpretation of in vitro modelling data, as discussed above, but also for the correlation of in vitro data with clinical phenotypes to uncover new prognostic biomarkers Conversely, re-use of clinical trial data on known patient biomarkers can inform the design of phenotypic assays in hiPSC-derived cells, and this generates more reliable modelling platforms Clearly, patient-level data need to be anonymized to protect patient privacy and avoid confidentiality violations.
This anonymization can be achieved through dataset de-identification and quality control, as well as data access control — users should be authorized and legally bound to data sharing agreements
Cell culture refers to the removal of cells from an animal or plant and their subsequent growth in a favorable artificial environment. The cells may be removed from the tissue directly and disaggregated by enzymatic or mechanical means before cultivation, or they may be derived from a cell line or cell strain that has already been established. Primary culture refers to the stage of the culture after the cells are isolated from the tissue and proliferated under the appropriate conditions until they occupy all of the available substrate i. At this stage, the cells have to be subcultured i. Cell lines derived from primary cultures have a limited life span i. A cell strain often acquires additional genetic changes subsequent to the initiation of the parent line.
The handbook and videos are intended as an introduction to cell culture basics, covering topics such other method, this cell line becomes a cell strain. A cell.
The Chemical Engineering Journal focuses upon three aspects of chemical engineering : chemical reaction engineering, environmental chemical engineering, and materials synthesis and processing. The Chemical Engineering Journal is an international research journal and invites contributions of original and novel fundamental research. The journal aims to provide an international forum for the presentation of original fundamental research, interpretative reviews and discussion of new developments in chemical engineering. Papers which describe novel theory and its application to practice are welcome, as are those which illustrate the transfer of techniques from other disciplines. Reports of carefully executed experimental work, which is soundly interpreted are also welcome.
From the ancient Romans, through the Middle Ages, to the late of the nineteenth century, the Aristotelian doctrine of spontaneous generation was one of the most basic laws. Even the invention of the microscope and investigations of Leeuwenhoek and Hook did not disprove the Aritostelian doctrine. Finally, in the eighteenth century, the spontaneous generation doctrine was laid by Louis Pasteur.
Cell culture refers to the removal of cells from an animal or plant and their subsequent growth in a favorable artificial environment. The cells may be removed from the tissue directly and disaggregated by enzymatic or mechanical means before cultivation, or they may be derived from a cell line or cell strain that has already been established. Primary culture refers to the stage of the culture after the cells are isolated from the tissue and proliferated under the appropriate conditions until they occupy all of the available substrate i.
Introduction To Density Worksheet Pdf. Put another way, density is the ratio between mass and volume or mass per unit volume. It could be used in a physical science, physics, or chemistry course depending on the student population. A gas with a volume of 4. Use a calculator to answer.
По изумлению на лице Чатрукьяна было видно, что он никогда прежде не бывал в этой комнате. Какова бы ни была причина его волнения, когда он колотил в стеклянную стену Третьего узла, она моментально улетучилась. Он разглядывал роскошную внутреннюю отделку, выстроившиеся в ряд компьютеры, диваны, книжные полки, залитые мягким светом. Увидав королеву шифровалки Сьюзан Флетчер, Чатрукьян моментально отвел. Он боялся ее как огня. Ее мозги работали словно на совсем другом уровне. Она подавляла его своей красотой, и всякий раз, когда он оказывался рядом, язык у него заплетался.
Необходимо было срочно что-то придумать. - Con permiso! - крикнул санитар. Мимо стремительно проплыла каталка. Беккер успел отскочить в сторону и окликнул санитара. - Dоnde esta el telefono.
Мы служба сопровождения, нас нечего стесняться. Красивые девушки, спутницы для обеда и приемов и все такое прочее.
В этот момент в тридцати метрах от них, как бы отвергая мерзкие признания Стратмора, ТРАНСТЕКСТ издал дикий, душераздирающий вопль. Звук был совершенно новым - глубинным, зловещим, нарастающим, похожим на змею, выползающую из бездонной шахты. Похоже, фреон не достиг нижней части корпуса. Коммандер отпустил Сьюзан и повернулся к своему детищу стоимостью два миллиарда долларов. Глаза его расширились от ужаса.
Это он должен был упасть замертво, а не бедолага азиат. - Клушар глотал ртом воздух, и Беккер начал волноваться. - Не знаете, как его зовут.
Она принялась нажимать кнопки безжизненной панели, затем, опустившись на колени, в отчаянии заколотила в дверь и тут же замерла. За дверью послышалось какое-то жужжание, словно кабина была на месте. Она снова начала нажимать кнопки и снова услышала за дверью этот же звук. И вдруг Сьюзан увидела, что кнопка вызова вовсе не мертва, а просто покрыта слоем черной сажи. Она вдруг начала светиться под кончиком пальца.
Tissue culture is the growth of tissues or cells in an artificial medium separate from the parent organism.AimГ© V. 01.06.2021 at 04:12
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Introduction to cell and tissue culture: theory and technique / The Practice and Theory of Tissue Culture. 3 Method for Fluorescent Detection of Mycoplasma.