We thank Dr. Nicolai Konow for providing the data used in this study. Funded by NSF IOS-2016049 and NSF DBI-2021832.

"Avatar"- Optimized Ex Vivo Work Loop Experiment to Emulate In Vivo Muscle Force

<ol>
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G., Heckman, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Doublet+potentiation+during+eccentric+and+concentric+contractions+of+cat+soleus+muscle.">Doublet potentiation during eccentric and concentric contractions of cat soleus muscle.</a> Journal of Applied Physiology. 82 (4), 1219-1228 (1997).</li><li>Marsh, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+muscles+deal+with+real-world+loads:+the+influence+of+length+trajectory+on+muscle+performance.">How muscles deal with real-world loads: the influence of length trajectory on muscle performance.</a> The Journal of Experimental Biology. 202, 3377-3385 (1999).</li><li>Hessel, A. L., Monroy, J. A., Nishikawa, K. 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All authors acknowledge there is no conflict of interest.

While organisms move seamlessly across landscapes, the underlying loads and strains that the muscles experience vary drastically1,6,23. During both in vivo locomotion1,24 and in &#34;avatar&#34; experiments, muscles are stimulated submaximally under cyclical, non-steady conditions. The isometric force-length and isotonic force-velocity relationships are not well...

Moving animals achieve impressive athletic feats of endurance, speed, and agility in complex environments. Animal locomotion is particularly impressive in contrast to human-engineered machines-the stability and agility of current-legged robots, prostheses, and exoskeletons remain poor compared to animals. Legged locomotion in natural terrain requires precise control and rapid adjustments to alter the speed and maneuver environmental features that act as unexpected perturbations1,2,3,4. Yet, understanding non-steady locomotion is inherently challenging because the dynamics depend on complex interactions between the physical environment, musculoskeletal mechanics, and sensorimotor control1,2. Legged locomotion requires responding to unexpected perturbations with rapid multi-modal processing of sensory information and coordinated actuation of limbs and joints1,5. Ultimately, movement is made possible by muscles producing force via intrinsic mechanical properties of the musculoskeletal system as well as from neural control1,5,6,7. An outstanding question of neuromechanics is how these factors interact to produce coordinated movement in response to unexpected perturbations. The following technique utilizes muscle&#39;s intrinsic mechanical response to deformation using in vivo strain trajectories during controllable ex vivo experiments with an &#34;avatar&#34; muscle.
The muscle work loop technique has provided an important framework for understanding intrinsic muscle mechanics during cyclical movements8,9,10. The traditional work loop technique drives muscles through predefined, typically sinusoidal, strain trajectories using frequencies and activation patterns measured during in vivo experiments2,8,9,11. Using sinusoidal length trajectories can realistically estimate work and power output during flight12 and swimming2 under conditions where animals do not undergo rapid changes in strain trajectories due to interaction with the environment and musculoskeletal kinematics. However, in vivo muscle strain trajectories during legged locomotion arise dynamically from interactions among neural activation, musculoskeletal kinematics, and loads applied by the environment5,7,13,14. A more realistic work loop technique is needed to emulate loads, strain trajectories, and force production that corresponds to in vivo muscle-tendon dynamics and provides insight into how intrinsic muscle mechanics and neural control interact to produce coordinated movement in the face of perturbations.
Here, we present a novel way to emulate in vivo muscle forces during treadmill locomotion by using an &#34;avatar&#34; muscle from a laboratory rodent during controlled ex vivo experiments with in vivo strain trajectories that represent time-varying in vivo loads. Using the measured in vivo strain trajectories from a target muscle on muscles from a laboratory animal during controlled ex vivo experiments will emulate loads experienced during locomotion. In the experiments described here, the ex vivo mouse extensor digitorum longus (EDL) muscle is used as an &#34;avatar&#34; for the in vivo rat medial gastrocnemius (MG) muscle during walking, trotting, and galloping on a treadmill13. This approach is similar to the work-loop technique, except that in vivo strain trajectories are scaled appropriately and imposed on ex vivo mouse muscles attached to a servo motor. While mouse EDL muscles differ in size, fiber type, and architecture compared to the rat MG, it is possible to control for these differences. The &#34;avatar&#34; technique allows one to: (1) emulate in vivo strain, activation, stride frequency, and work-loop patterns; (2) vary these patterns to match in vivo force responses most accurately; and (3) vary specific features of strain and/or activation in controlled combinations to test mechanistic hypotheses.

<table><tbody><tr><td>Braided Non-Absorbable Silk Suture 4-0&amp;nbsp;</td><td>Mersilk&amp;nbsp;</td><td>734H</td><td></td></tr><tr><td>Calcium Chloride Dihydrate (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>1086436</td><td>Krebs-Henseleit&amp;nbsp;solution</td></tr><tr><td>Dextrose&amp;nbsp;</td><td>Sigma-Aldrich</td><td>D9434</td><td>Krebs-Henseleit&amp;nbsp;solution</td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>PHR1428</td><td>Krebs-Henseleit&amp;nbsp;solution</td></tr><tr><td>Hydorchloric Acid (HCl)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>1.37055</td><td>Krebs-Henseleit&amp;nbsp;solution</td></tr><tr><td>LabView Data Collection&amp;nbsp;</td><td>Lab-View</td><td></td><td></td></tr><tr><td>Magnesium Sulfate (MgSO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>M7506</td><td>Krebs-Henseleit&amp;nbsp;solution</td></tr><tr><td>Potassium Chloride (KCl)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P3911</td><td>Krebs-Henseleit&amp;nbsp;solution</td></tr><tr><td>Potassium Phosphate Monobasic (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>5.43841</td><td>Krebs-Henseleit&amp;nbsp;solution</td></tr><tr><td>S88 Stimulator</td><td>Grass</td><td>M643H05</td><td>Available for purchase on Ebay</td></tr><tr><td>Series 300B Lever System</td><td>Aurora</td><td>1200A</td><td>includes water-jacket tissue bath</td></tr><tr><td>Sodium Bicarbonate (NaHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>S5761</td><td>Krebs-Henseleit&amp;nbsp;solution</td></tr><tr><td>Sodium Chloride (NaCl)&amp;nbsp;</td><td>Sigma-Aldrich</td><td>S9888</td><td>Krebs-Henseleit&amp;nbsp;solution</td></tr><tr><td>Sodium Hydroxide (NaOH)</td><td>Sigma-Aldrich</td><td>S5881</td><td>Krebs-Henseleit&amp;nbsp;solution</td></tr><tr><td>Wild Type Mice</td><td>Jackson Laboratory</td><td>B6C3Fe a/a Ttn mdm/J</td><td></td></tr></tbody></table>

"avatar", a modified ex vivo work loop experiments using in vivo strain and activation

Movement behaviors are emergent features of dynamic systems that result from muscle force production and work output. The interplay between neural and mechanical systems occurs at all levels of biological organization concurrently, from the tuning of leg muscle properties while running to the dynamics of the limbs interacting with the ground. Understanding the conditions under which animals shift their neural control strategies toward intrinsic muscle mechanics (&#39;preflexes&#39;) in the control hierarchy would allow muscle models to predict in vivo muscle force and work more accurately. To understand in vivo muscle mechanics, ex vivo investigation of muscle force and work under dynamically varying strain and loading conditions similar to in vivo locomotion is required. In vivo strain trajectories typically exhibit abrupt changes (i.e., strain and velocity transients) that arise from interactions among neural activation, musculoskeletal kinematics, and loads applied by the environment. The principal goal of our &#34;avatar&#34; technique is to investigate how muscles function during abrupt changes in strain rate and loading when the contribution of intrinsic mechanical properties to muscle force production may be highest. In the &#34;avatar&#34; technique, the traditional work-loop approach is modified using measured in vivo strain trajectories and electromyographic (EMG) signals from animals during dynamic movements to drive ex vivo muscles through multiple stretch-shortening cycles. This approach is similar to the work-loop technique, except that in vivo strain trajectories are scaled appropriately and imposed on ex vivo mouse muscles attached to a servo motor. This technique allows one to: (1) emulate in vivo strain, activation, stride frequency, and work-loop patterns; (2) vary these patterns to match in vivo force responses most accurately; and (3) vary specific features of strain and/or activation in controlled combinations to test mechanistic hypotheses.

The goal of the &#34;avatar&#34; experiments is to replicate in vivo force production and work output as closely as possible during ex vivo work loop experiments. This study chose to use mouse EDL as an &#34;avatar&#34; for rat MG because mouse EDL and rat MG are both comprised of mostly of fast-twitch muscles20,21. Both muscles are primary movers of the ankle joint (EDL ankle dorsiflexor, MG ankle plantarflexor) with similar pennation angles (m...

Watch this Scientific Journal Video about Bridging the Gap Between In Vivo and Ex Vivo Studies with the "Avatar" Technique to Advance Muscle Mechanics Research at JoVE.com

Author Spotlight: Bridging the Gap Between In Vivo and Ex Vivo Studies with the "Avatar" Technique to Advance Muscle Mechanics Research 

This article details the methodology for emulating in vivo muscle force production during ex vivo work loop experiments using an "avatar" muscle from a laboratory rodent to assess the contributions of strain transients and activation to the muscle force response.

"Avatar", a Modified Ex vivo Work Loop Experiments Using In vivo Strain and Activation

Movement behaviors are emergent features of dynamic systems that result from muscle force production and work output. The interplay between neural and ...

avatar-modified-ex-vivo-work-loop-experiments-using-vivo-strain

Research

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Bioengineering

708 Views.  Northern Arizona University. This article details the methodology for emulating in vivo muscle force production during ex vivo work loop experiments using an "avatar" muscle from a laboratory rodent to assess the contributions of strain transients and activation to the muscle force response. 

Video: Author Spotlight: Bridging the Gap Between In Vivo and Ex Vivo Studies with the "Avatar" Technique to Advance Muscle Mechanics Research 

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Ex Vivo Culture of Patient Tissue & Examination of Gene Delivery

This article describes the culture of patient tissue slices for gene delivery studies and subsequent analysis of gene expression using IVIS bioluminescence imaging.

This video describes the use of patient tissue as an ex vivo model for the study of gene delivery.  Fresh patient tissue obtained at the time of surgery ...

Medicine

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the gastrointestinal tract, as resident microbiota occurs and prevents colonisation by exogenous bacteria. Indeed, more than 600 species of bacteria are found in the oral cavity, and a single individual may carry around 100 different at any time. Oral bacteria possess the ability to adhere to the various niches in the oral ecosystem, thus becoming integrated within the resident microbial communities, and favouring growth and survival. However, the flow of bacteria into the gut during swallowing has been proposed to disturb the balance of the gut microbiota. In fact, oral administration of P. gingivalis shifted bacterial composition in the ileal microflora. We used a synthetic community as a simplified representation of the natural oral ecosystem, to elucidate the survival and viability of oral bacteria subjected to simulated gastrointestinal transit conditions. Fourteen species were selected, subjected to in vitro salivary, gastric, and intestinal digestion processes, and presented to a multicompartment cell model containing Caco-2 and HT29-MTX cells to simulate the gut mucosal epithelium. This model served to unravel the impact of swallowed bacteria on cells involved in the enterohepatic circulation. Using synthetic communities allows for controllability and reproducibility. Thus, this methodology can be adapted to assess pathogen viability and subsequent inflammation-associated changes, colonization capacity of probiotic mixtures, and ultimately, potential bacterial impact on the presystemic circulation.

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

Gut host-microbe interactions were assessed using a novel approach combining a synthetic oral community, in vitro gastrointestinal digestion, and a model of the small intestine epithelium. We present a method that can be adapted to evaluate cell invasion of pathogens and multi-species biofilms, or even to test probiotic formulations&#39; survivability.

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the ...

Biochemistry

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the complexities of the organoid growth characteristics carry significant caveats for the investigator. Specifically, the growth patterns of each individual organoid are highly variable and create a heterogeneous population of epithelial cells in culture. With such caveats, common tissue culture practices cannot be simply applied to the organoid system due to the complexity of the cellular structure. Counting and plating based solely on cell number, which is common for individually separated cells, such as cell lines, is not a reliable method for organoids unless some normalization technique is applied. Normalizing to total protein content is made complex due to the resident protein matrix. These characteristics in terms of cell number, shape and cell type should be taken into consideration when evaluating secreted contents from the organoid mass. This protocol has been generated to outline a simple procedure to culture and treat small intestinal organoids with microbial pathogens and pathogen associated molecular patterns (PAMPs). It also emphasizes the normalization techniques that should be applied when protein analysis are conducted after such a challenge.

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Here, a protocol to harvest, maintain, and treat mouse small intestinal organoids with pathogen associated molecular patterns (PAMPs) and Listeria monocytogenes is described, as well as emphasis on gene expression and proper normalization techniques for protein.

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the ...

Immunology and Infection

The Ex Vivo Colon Organ Culture and Its Use in Antimicrobial Host Defense Studies

The intestine displays an architecture of repetitive crypt structures consisting of different types of epithelial cells, lamina propia containing immune cells, and stroma. All of these heterogeneous cells contribute to intestinal homeostasis and participate in antimicrobial host defense. Therefore, identifying a surrogate model for studying immune response and antimicrobial activity of the intestine in an in vitro setting is extremely challenging. In vitro studies using immortalized intestinal epithelial cell lines or even primary crypt organoid culture do not represent the exact physiology of normal intestine and its microenvironment. Here, we discuss a method of culturing mouse colon tissue in a culture dish and how this ex vivo organ culture system can be implemented in studies related to antimicrobial host defense responses. In representative experiments, we showed that colons in organ culture express antimicrobial peptides in response to exogenous IL-1&#946; and IL-18. Further, the antimicrobial effector molecules produced by the colon tissues in the organ culture efficiently kill Escherichia coli in vitro. This approach, therefore, can be utilized to dissect the role of pathogen- and danger-associated molecular patterns and their cellular receptors in regulating intestinal innate immune responses and antimicrobial host defense responses.

The Ex Vivo Colon Organ Culture and Its Use in Antimicrobial Host Defense Studies

The ex vivo organ culture allows investigation of biological processes in the context of the intact tissue architecture. Here, we introduce a method of ex vivo culture of the mouse colon, which can be used to study innate immunity and antimicrobial host defense in the intestine.

The intestine displays an architecture of repetitive crypt structures consisting of different types of epithelial cells, lamina propia containing immune ...

Adult Mouse DRG Explant and Dissociated Cell Models to Investigate Neuroplasticity and Responses to Environmental Insults Including Viral Infection

This protocol describes an ex vivo model of mouse-derived dorsal root ganglia (DRG) explant and in vitro DRG-derived co-culture of dissociated sensory neurons and glial satellite cells. These are useful and versatile models to investigate a variety of biological responses associated with physiological and pathological conditions of the peripheral nervous system (PNS) ranging from neuron-glial interaction, neuroplasticity, neuroinflammation, and viral infection. The usage of DRG explant is scientifically advantageous compared to simplistic single cells models for multiple reasons. For instance, as an organotypic culture, the DRG explant allows ex vivo transfer of an entire neuronal network including the extracellular microenvironment that play a significant role in all the neuronal and glial functions. Further, DRG explants can also be maintained ex vivo for several days and the culture conditions can be perturbed as desired. In addition, the harvested DRG can be further dissociated into an in vitro co-culture of primary sensory neurons and satellite glial cells to investigate neuronal-glial interaction, neuritogenesis, axonal cone interaction with the extracellular microenvironment, and more general, any aspect associated with the neuronal metabolism. Therefore, the DRG-explant system offers a great deal of flexibility to study a wide array of events related to biological, physiological, and pathological conditions in a cost-effective manner.

In this report, the advantages of organotypic cultures and dissociated primary cultures of mouse-derived dorsal root ganglia are highlighted to investigate a wide range of mechanisms associated with neuron-glial interaction, neuroplasticity, neuroinflammation, and response to viral infection.

This protocol describes an ex vivo model of mouse-derived dorsal root ganglia (DRG) explant and in vitro DRG-derived co-culture of dissociated sensory ...

Neuroscience

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca2+ handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca2+ signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e., mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle.

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca2+ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.

 
Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, ...

Biology

Fabricating Mouse Tendon Assembloids to Study Cellular Interactions in Disease

Engineering Tendon Assembloids to Probe Cellular Crosstalk in Disease and Repair

Tendons enable locomotion by transferring muscle forces to bones. They rely on a tough tendon core comprising collagen fibers and stromal cell populations. This load-bearing core is encompassed, nourished, and repaired by a synovial-like tissue layer comprising the extrinsic tendon compartment. Despite this sophisticated design, tendon injuries are common, and clinical treatment still relies on physiotherapy and surgery. The limitations of available experimental model systems have slowed the development of novel disease-modifying treatments and relapse-preventing clinical regimes. 
In vivo human studies are limited to comparing healthy tendons to end-stage diseased or ruptured tissues sampled during repair surgery and do not allow the longitudinal study of the underlying tendon disease. In vivo animal models also present important limits regarding opaque physiological complexity, the ethical burden on the animals, and large economic costs associated with their use. Further, in vivo animal models are poorly suited to systematic probing of drugs and multicellular, multi-tissue interaction pathways. Simpler in vitro model systems have also fallen short. One major reason is a failure to adequately replicate the three-dimensional mechanical loading necessary to meaningfully study tendon cells and their function. 
The new 3D model system presented here alleviates some of these issues by exploiting murine tail tendon core explants. Importantly, these explants are easily accessible in large numbers from a single mouse, retain 3D in situ loading patterns at the cellular level, and feature an in vivo-like extracellular matrix. In this protocol, step-by-step instructions are given on how to augment tendon core explants with collagen hydrogels laden with muscle-derived endothelial cells, tendon-derived fibroblasts, and bone marrow-derived macrophages to substitute disease- and injury-activated cell populations within the extrinsic tendon compartment. It is demonstrated how the resulting tendon assembloids can be challenged mechanically or through defined microenvironmental stimuli to investigate emerging multicellular crosstalk during disease and injury.

Author Spotlight: Advancing Tendon Research by Developing Mouse Assembloids to Understand Cellular Mechanisms

Here, we present an assembloid model system to mimic tendon cellular crosstalk between the load-bearing tendon core tissue and an extrinsic compartment containing cell populations activated by disease and injury. As an important use case, we demonstrate how the system can be deployed to probe disease-relevant activation of extrinsic endothelial cells.

Tendons enable locomotion by transferring muscle forces to bones. They rely on a tough tendon core comprising collagen fibers and stromal cell ...

A Novel High-Throughput Ex Vivo Ovine Skin Wound Model for Testing Emerging Antibiotics

The development of antimicrobials is an expensive process with increasingly low success rates, which makes further investment in antimicrobial discovery research less attractive. Antimicrobial drug discovery and subsequent commercialization can be made more lucrative if a fail-fast-and-fail-cheap approach can be implemented within the lead optimization stages where researchers have greater control over drug design and formulation. In this article, the setup of an ex vivo ovine wounded skin model infected with Staphylococcus aureus&#160;is described, which is simple, cost-effective, high throughput, and reproducible. The bacterial physiology in the model mimics that during infection as bacterial proliferation is dependent on the pathogen&#39;s ability to damage the tissue. The establishment of wound infection is verified by an increase in viable bacterial counts compared to the inoculum. This model can be used as a platform to test the efficacy of emerging antimicrobials in the lead optimization stage. It can be contended that the availability of this model will provide researchers developing antimicrobials with a fail-fast-and-fail-cheap model, which will help increase success rates in subsequent animal trials. The model will also facilitate the reduction and refinement of animal use for research and ultimately enable faster and more cost-effective translation of novel antimicrobials for skin and soft tissue infections to the clinic.

A Novel High-Throughput Ex Vivo Ovine Skin Wound Model for Testing Emerging Antibiotics

The protocol describes a step-by-step method to set up an ex vivo ovine wounded skin model infected with Staphylococcus aureus. This high-throughput model better simulates infections in vivo compared with conventional microbiology techniques and presents researchers with a physiologically relevant platform to test the efficacy of emerging antimicrobials.

The development of antimicrobials is an expensive process with increasingly low success rates, which makes further investment in antimicrobial discovery ...

Single Myofiber Culture Assay for the Assessment of Adult Muscle Stem Cell Functionality Ex Vivo

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells leave their quiescent state and activate the myogenic program ultimately leading to the repair of damaged tissue concomitant with the replenishment of the muscle stem cell pool. Various factors influence muscle stem cell activity, among them intrinsic stimuli but also signals from the direct muscle stem cell environment, the stem cell niche. The isolation and culture of single myofibers with their associated muscle stem cells preserves most of the interaction of the stem cell with its niche and is, therefore, the closest possibility to study muscle stem cell functionality ex vivo. Here, a protocol for the isolation, culture, siRNA transfection and immunostaining of muscle stem cells on their respective myofibers from mouse EDL (extensor digitorum longus) muscles is provided. The experimental conditions outlined here allow the study and manipulation of muscle stem cells ex vivo including investigation of myogenic activity without the inherent need for in vivo animal experiments.

In this protocol an in vitro culturing and functional analysis method for muscle stem cells is described, which preserves most of their interactions with their endogenous niche.

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells ...

Ex Vivo Expansion and Genetic Manipulation of Mouse Hematopoietic Stem Cells in Polyvinyl Alcohol-Based Cultures

Self-renewing&#160;multipotent&#160;hematopoietic stem cells (HSCs) are an important cell type due to their abilities to support hematopoiesis throughout life and reconstitute the entire blood system following transplantation. HSCs are used clinically in stem cell transplantation therapies, which represent curative treatment for a range of blood diseases. There is substantial interest in both understanding the mechanisms that regulate HSC activity and hematopoiesis, and developing new HSC-based therapies. However, the stable culture and expansion of HSCs ex vivo has been a major barrier in studying these stem cells in a tractable ex vivo system. We recently developed a polyvinyl alcohol-based culture system that can support the long-term and large-scale expansion of transplantable mouse HSCs and methods to genetically edit them. This protocol describes methods to culture and genetically manipulate mouse HSCs via electroporation and lentiviral transduction. This protocol is expected to be useful to a wide range of experimental hematologists interested in HSC biology and hematopoiesis.

Ex Vivo Expansion and Genetic Manipulation of Mouse Hematopoietic Stem Cells in Polyvinyl Alcohol-Based Cultures

Presented here is a protocol to initiate, maintain, and analyze mouse hematopoietic stem cell cultures using ex vivo polyvinyl alcohol-based expansion, as well as methods to genetically manipulate them by lentiviral transduction and electroporation.

Self-renewing&#160;multipotent&#160;hematopoietic stem cells (HSCs) are an important cell type due to their abilities to support hematopoiesis throughout ...

"Avatar", a Modified Ex vivo Work Loop Experiments Using In vivo Strain and Activation

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