Stem Cell Culture And Its Application Pdf


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Stem cells are the foundation for every organ and tissue in your body. There are many different types of stem cells that come from different places in the body or are formed at different times in our lives. These include embryonic stem cells that exist only at the earliest stages of development and various types of tissue-specific or adult stem cells that appear during fetal development and remain in our bodies throughout life. Beyond these two critical abilities, though, stem cells vary widely in what they can and cannot do and in the circumstances under which they can and cannot do certain things.

Stem Cell Engineering

Thank you for visiting nature. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser or turn off compatibility mode in Internet Explorer. In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. We constructed an automated cell culture platform optimised for long-term maintenance and monitoring of different cells in three dimensional microfluidic cell culture devices.

The system can be flexibly adapted to various experimental protocols and features time-lapse imaging microscopy for quality control and electrophysiology monitoring to assess cellular activity. Calcium imaging confirmed the electrophysiological activity of differentiated neurons and immunostaining confirmed the efficiency of the differentiation protocol.

This system is the first example of an automated Organ-on-a-Chip culture and has the potential to enable a versatile array of in vitro experiments for patient-specific disease modelling.

Laboratory automation is becoming increasingly prevalent in the life sciences 1 , 2. Automated cell culture has the potential to increase the quantity and the quality of experiments that can be completed in parallel and enables long-term cell culture maintenance with reduced manual labour 3. Once an automated protocol is established, a robot can operate continuously without fatigue and with the same consistency and accuracy 2.

Likewise, once established an automated imaging system can take repeated measurements over a long period without intervention 4. The combination of robotic cell culture and automated imaging has a wide range of biological applications. A leading example is their use to distinguish causation from correlation in the pathogenesis of neurodegenerative diseases by longitudinal measurement of human in vitro disease models 5.

Laboratory automation requires precise specification of, and enables fine control over, many experimental protocol parameters, such as dispensing speed, cell culture conditions, fluid temperature and measurements. This enhances experimental reproducibility by reducing variance between replicates 6.

In vitro cell culture automation facilitates faithful replication of certain in vivo physiological conditions as it enables quantitative control over key experimental parameters, e. This increases the validity of employing an in vitro model to represent an in vivo system, in health or disease, thereby accelerating biomedical research. During manual cell culture, procedures involving liquid handling, such as dispensing media, aspiring media, and movement of liquid samples between containers, are essential to all protocols.

Therefore, when a cell culture protocol is automated, a liquid-handler and a robot for transposition of receptacles, are two of the most important devices. There are two types of technologies used in liquid-handler: contact and non-contact dispensing 2.

On one hand, to dispense a precise volume, contact dispensing requires the head of the tip holding the fluid to touch the bottom of the substrate; for instance, the bottom of a well or to touch the liquid surface.

On the other hand, non-contact dispensing does not require any contact between the tip and the substrate or liquid surface for liquid release. Dispensing can require the handling of very small volumes, as low as a few nano-litres, so the technological advances in liquid handlers have focused more on dispensing than on aspiration 2.

Low volume dispensing and aspiration are especially required for microfluidic cell culture 7. The robot to move receptacles can be a robotic arm or a gantry robot with a gripper for receptacles 2. A gantry robot only moves in Cartesian coordinates, where the three principal axes of control have linear actuators.

The choice of devices used in laboratory automation should be based on their intended uses, flexibility, purchase costs and maintenance costs. Selecting the components of an automated plant usually entails having to purchase devices from different manufacturers, as no single firm supplies all of the devices that might be required to automate a laboratory protocol.

Therefore, all of the components must be amenable to software integration in order to be able to function as a single autonomous plant.

Computer scripting achieves integration by assigning a master software that communicates directly with all devices 8. In this approach, assuming that all the devices are able to send and receive commands, a communication protocol must be implemented that is compatible with each individual device.

However, this approach requires the master device software to recognise every other device using an idiosyncratic communication protocol. This approach can be very expensive and challenging to implement. Furthermore, SiLA defines over 30 standard device classes used in the field of life sciences, including incubators, microscopes, de-lidders and liquid handlers 9.

For each device class, a list of required and optional functions are proposed to standardise the software communication within a laboratory automation plant. This approach standardises the communication between all of the devices of a plant, regardless of the manufacturer, and a SiLA compatible process management software can then be used to control each SiLA compatible device, without any modification.

These neuronal losses include cholinergic neurons, noradrenergic neurons and dopaminergic neurons which play a critical role in brain function by releasing a neurotransmitter called dopamine 12 , 13 , 14 , 15 , Reinhardt et al. Microfluidic cell culture concerns the design and implementation of devices and protocols for the culture, maintenance and perturbation of cells in micro-scale fluid volumes.

The reasons behind the popularity of microfluidic cell culture are both economic and scientific. Cell culture reagents are expensive, and the amounts used in microfluidic cell cultures are much less than in macroscopic cell culture 21 , Microfluidic cell culture also has the potential to lower the ratio of extracellular to intracellular fluid volumes, thereby decreasing the temporal lag in extracellular response to molecules transported across cell membranes, e.

With the advent of Organ-on-a-Chip technology 26 , microfluidic cell culture has developed tremendously and includes examples of perfusion culture, co-culture and three dimensional cell cultures 27 , 28 , Moreover, miniaturisation enables multiple experimental replicates within a geometrically confined experimental footprint. Thus far, no examples are known of an Organ-on-a-Chip operation in an automated setting, although few hold the promise to do so Even though the combination of automation, microfluidics and cell culture technologies allows the screening of multiple environmental conditions in parallel 31 , 32 , as well as enabling regular live cell culture monitoring 33 , 34 at a temporal resolution impractically in a manual setting.

Therefore, laboratory automation technology is key to unleash the full potential of microfluidic cell culture. Subsequently, we implemented the differentiation of hNESCs into three dimensional networks of electrophysiologically active dopaminergic neurons into the OrganoPlate The microfluidic titer plate was designed for compatibility with laboratory automation, but this has yet to be exploited.

The manual culture of human pluripotent stem cell derived cells within the microfluidic titer plate has also been established, but the potential for automation has also not yet been exploited.

Herein, we report the integration of developmental biology, microfluidic cell culture and laboratory automation technology to generate a flexible automated, enclosed microfluidic and macroscopic cell culture observatory, termed the Pelican. We elaborate on each device in the Pelican, as well as the SiLA software integration approach used to realise an automated system.

We illustrate the functionality of the Pelican for automated cell culture and differentiation of human neuroepithelial stem cells into dopaminergic neurons, within a three-dimensional microfluidic device We monitored the health of the cells throughout the experiment with an automated image acquisition pipeline.

After 24 days in culture, we assessed the outcome by characterising known features of dopaminergic neurons by calcium imaging and immunofluorescence assays. Three dimensional imaging revealed mature and interconnected neuronal populations within microfluidic cell culture chips.

The Pelican is a modular automation system, compatible with implementation of a variety of automation platforms, where cost-effective flexibility is maximised to allow for replacement or further expansion of platforms by integration of new devices. Microfluidic cell culture has already been manually integrated with iPSC technology Our work integrates an automated system with an Organ-on-a-Chip stem cell culture.

The different devices and their use in the automated system are detailed in the Supplementary Experimental Procedures. Pelican automated cell culture observatory. A Top view inside the Pelican automation workstation without housing: 1 Wide angle lens image of the automated enclosure. B Outside view of automated culture system top. Front bottom left and rear bottom right views of Pelican with housing. Yellow imaging station , light blue liquid handling station , green level of stainless steel work surface and orange waste containers.

The colour codes of the devices labels in A and Fig. S1 match. The robot was attached to the top of the stainless steel frame support inside the housing through rails that allow the movement of the robotic arm along three axes. This maximised the modular capacity of the Pelican because the rails determine the robotically useful space of the system, in contrast to other automated systems with a fixed rotating robot arm, which often has limited reach.

The useful space is the space that is available to potentially hold new devices, adding to the functions of the automated platform. In addition, a liquid dispenser with only non-contact dispensing was also implemented in the system. The liquid dispenser is less precise than the liquid-handler. However, it is much faster as it can handle up to 96 wells per step compared to 4 wells per step for the pipetting modules. Despite its shortcomings, the contact dispensing function of the ZEUS is especially useful for microfluidic cell culture where very small volumes must be dispensed.

In addition, the contact dispensing helps to make sure that the dispensed media is bubble free. This is very important as with low flow rate non-contact dispensing, bubbles that arise can imped the flow of fresh media, which could ultimately starve the cells in a chip. Contact dispensing is very precise for dispensing small volumes. However, this precision is dependent on dispensing at an exact location, which is not always possible as the dispensing tip cannot always physically access the well to make contact with the liquid 1.

With contact dispensing, well cross-contamination is a risk as there is direct contact between the tip and the liquid in the destination well. Therefore, a cleaning protocol was implemented after each dispensing step.

This does not promote speed and high-throughput capabilities so sought after in laboratory automation, however, the contact dispensing was only utilised for the initial loading of media to avoid the introduction of bubbles in the dry medium lane.

Non-contact dispensing does not require any contact between the dispensing tip and the liquid. This helps to avoid cross-contamination, and promotes the integrity of the well. Non-contact dispensing is very popular in laboratory automation because it is versatile, and it is easy to dispense to any area of a well regardless of geometry such as undercuts, so long as there is an opening on the well As a result, the non-contact dispensing methodology was utilised for all subsequent media changes.

The SiLA standard was consistently implemented for networking and integrating the devices and components. In brief, LACS is a programming environment for laboratory automation. The running of all existing protocols through LACS GUI merely requires the identification of the required protocol and the input of the required plate name by the operator.

LACS does not make any process-specific decisions, nor does it analyse any data. In the occurrence of an unexpected event a device failure or any other error , the operator is always prompted to assess and rectify the error or the event.

KG, Germany. A digital and analogue logic device has a binary set; a binary input and binary output. A single weidmuller SiLA driver was installed to control all devices connected to one logic module.

In the first case it holds the information, which position is occupied and with which substrate and which free position could potentially hold which type of substrate. The software knows each position and status as well as each substrate in the plant including position and type.

Like the hardware devices, this virtual device has a SiLA communication interface and a Windows 7 graphical user interface to view the stored data. The integration of new devices requires two or three steps depending on the complexity of the device and the availability of compatible drivers. The first step requires the generation of a driver for the new devices.

Automated microfluidic cell culture of stem cell derived dopaminergic neurons

Thank you for visiting nature. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser or turn off compatibility mode in Internet Explorer. In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. We constructed an automated cell culture platform optimised for long-term maintenance and monitoring of different cells in three dimensional microfluidic cell culture devices. The system can be flexibly adapted to various experimental protocols and features time-lapse imaging microscopy for quality control and electrophysiology monitoring to assess cellular activity.

Metrics details. In recent years, stem cell therapy has become a very promising and advanced scientific research topic. The development of treatment methods has evoked great expectations. This paper is a review focused on the discovery of different stem cells and the potential therapies based on these cells. The genesis of stem cells is followed by laboratory steps of controlled stem cell culturing and derivation.

Embryonic stem cells ES cells or ESCs are pluripotent stem cells derived from the inner cell mass of a blastocyst , an early-stage pre- implantation embryo. Isolating the embryoblast , or inner cell mass ICM results in destruction of the blastocyst, a process which raises ethical issues , including whether or not embryos at the pre-implantation stage should have the same moral considerations as embryos in the post-implantation stage of development. Researchers are currently focusing heavily on the therapeutic potential of embryonic stem cells, with clinical use being the goal for many laboratories. However, adverse effects in the research and clinical processes such as tumours and unwanted immune responses have also been reported. Embryonic stem cells ESCs , derived from the blastocyst stage of early mammalian embryos, are distinguished by their ability to differentiate into any embryonic cell type and by their ability to self-renew. It is these traits that makes them valuable in the scientific and medical fields. ESCs have a normal karyotype , maintain high telomerase activity, and exhibit remarkable long-term proliferative potential.


through cell culture. Highly plastic adult stem cells from a variety of sources, including umbilical cord blood and bone marrow, are routinely used in medical.


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Human pluripotent stem cells hPSCs are important resources for cell-based therapies and pharmaceutical applications. In order to realize the potential of hPSCs, it is critical to develop suitable technologies required for specific applications. Most hPSC technologies depend on cell culture, and are critically influenced by culture medium composition, extracellular matrices, handling methods, and culture platforms. This review summarizes the major technological advances in hPSC culture, and highlights the opportunities and challenges in future therapeutic applications. Core tip: This review summarizes recent developments in cell culture systems for human pluripotent stem cells, including signal transduction requirements at different pluripotency stages, advances in extracellular matrices and handling methods, establishment of chemically defined conditions, and various cell culture platforms for specific purposes.

We all have stem cells at work inside us. Right now, inside your bone marrow, stem cells are busy making the , million new blood cells you need every single day! We need to make new cells all the time, just to keep our body functioning. Some specialized cells, such as blood and muscle cells, are unable to make copies of themselves through cell division. Instead they are replenished from populations of stem cells.

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Stem cells: past, present, and future

Most cells in the body are differentiated cells. These cells can only serve a specific purpose in a particular organ. For example, red blood cells are specifically designed to carry oxygen through the blood. All humans start out as only one cell. This cell is called a zygote, or a fertilized egg. The zygote divides into two cells, then four cells, and so on. Eventually, the cells begin to differentiate, taking on a certain function in a part of the body.

Most cells in the body are differentiated cells. These cells can only serve a specific purpose in a particular organ. For example, red blood cells are specifically designed to carry oxygen through the blood. All humans start out as only one cell. This cell is called a zygote, or a fertilized egg. The zygote divides into two cells, then four cells, and so on. Eventually, the cells begin to differentiate, taking on a certain function in a part of the body.

Principles and Applications

Human embryonic stem cells hESCs hold great potential for the treatment of various degenerative diseases. Pluripotent hESCs have a great ability to undergo unlimited self-renewal in culture and to differentiate into all cell types in the body. The journey of hESC research is not that smooth, as it has faced several challenges which are limited to not only tumor formation and immunorejection but also social, ethical, and political aspects. The isolation of hESCs from the human embryo is considered highly objectionable as it requires the destruction of the human embryo. The issue was debated and discussed in both public and government platforms, which led to banning of hESC research in many countries around the world. The banning has negatively affected the progress of hESC research as many federal governments around the world stopped research funding. Afterward, some countries lifted the ban and allowed the funding in hESC research, but the damage has already been done on the progress of research.

Cell culture is the process by which cells are grown under controlled conditions, generally outside their natural environment. After the cells of interest have been isolated from living tissue , they can subsequently be maintained under carefully controlled conditions. These conditions vary for each cell type, but generally consist of a suitable vessel with a substrate or medium that supplies the essential nutrients amino acids , carbohydrates , vitamins , minerals , growth factors , hormones , and gases CO 2 , O 2 , and regulates the physio-chemical environment pH buffer , osmotic pressure , temperature. Most cells require a surface or an artificial substrate adherent or monolayer culture whereas others can be grown free floating in culture medium suspension culture. In practice, the term "cell culture" now refers to the culturing of cells derived from multicellular eukaryotes , especially animal cells, in contrast with other types of culture that also grow cells, such as plant tissue culture , fungal culture, and microbiological culture of microbes.

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