Help us improve our products. Sign up to take part. A Nature Research Journal. The development of laboratory-grown tissues, referred to as organoids, bio-artificial tissue or tissue-engineered constructs, is clearly expanding. We describe for the first time how engineered human muscles can be applied as a pre- or non-clinical model for intramuscular drug injection to further decrease and complement the use of in vivo animal studies.
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The human bio-artificial muscle BAM is formed in a seven day tissue engineering procedure during which human myoblasts fuse and differentiate to aligned myofibers in an extracellular matrix. The dimensions of the BAM constructs allow for injection and follow-up during several days after injection.
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A stereotactic setup allows controllable injection at multiple sites in the BAM. We injected several compounds; a dye, a hydrolysable compound, a reducible substrate and a wasp venom toxin. Afterwards, direct reflux, release and metabolism were assessed in the BAM constructs in comparison to 2D cell culture and isolated human muscle strips. Spectrophotometry and luminescence allowed to measure the release of the injected compounds and their metabolites over time. We also determined compound toxicity on the BAMs by measuring creatine kinase release in the medium, which increased with increasing toxic insult.
Taken together, we show that the BAM is an injectable human 3D cell culture model that can be used to measure release and metabolism of injected compounds in vitro.
When testing drug candidates non-clinically, adequate model systems are crucial for a high success rate in clinical trials. Currently, 2D cell culture models are widespread as one of the model systems for high-throughput screening since they are very accessible. However, they lack the natural cell environment. Moreover in 2D cell culture, compounds added to the cell culture medium can easily access the target cells and are exposed homogenously to all cells, in contrast to drug exposure to tissues in vivo 5 , 6.
These limitations are well-known and therefore the next step is drug testing in animal models. This allows evaluation of the route of administration, pharmacokinetics and pharmacodynamics. Major drawbacks are the cost of animal experiments and the fact that animal physiology differs from human physiology. These issues have led to a growing interest in creating 3D non-clinical human tissue models using tissue engineering.
In tissue engineering, cells are used with natural or synthetic scaffolds to create in vitro bio-artificial tissue that can replace in vivo tissue in various applications Three-dimensional tissue can also be created by differentiation from stem cells.
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Tissues derived by orchestrated in vitro stem cell differentiation are referred to as organoids. Different tissues such as cartilage, bone, heart and skeletal muscle, skin, liver and nerve are being developed 12 , 13 , 14 , 15 , 16 , 17 , Most of the approaches are still in research phase, but some products are already used in clinical practice. For example skin replacement products are already used in burn wound treatment reviewed in Besides the use of human in vitro grown tissues in regenerative medicine, they can also be used as models for disease aetiology, study of development or for drug testing purposes For example, commercially available hepatocyte and skin tissue models were described to more accurately reflect the human in vivo physiology and have a more representative drug response than 2D cell culture models 21 , Despite the increased interest, many tissue engineering hurdles remain to be tackled.
Most of the in vitro tissues have relatively small dimensions often submillimeter and when used for drug testing they are incubated in a medium containing the drug.
Other injection routes include subcutaneous, intravenous or intradermal Intramuscular injections deliver medication deep into the muscles, serving as a depot, allowing the medication to be gradually released in the blood. When looking at the onset of effect after administration, IV injections produce the most rapid onset of effect, followed by IM and SC injections So, although the IM route administration is not the fastest way to deliver a drug, it is preferred for drugs that require a longer efficacy.
The injection creates a drug depot in the muscle tissue and the drug is released slowly over a certain period of time For example, longer term low plasma levels were found for the IM administrated antipsychotic drug haloperidol decanoate in contrast to orally administrated haloperidol in schizophrenia patients, limiting side effects 26 , Due to lack of alternatives, candidate drugs and drug formulations for intramuscular administration are tested using methods developed for oral drug administration.
In vitro dissolution testing of candidate drugs is used to predict in vivo drug release profiles 28 , 29 , 30 , But in vitro results obtained from these studies cannot adequately predict in vivo behavior of intramuscular drug formulations, due to a lack of good representation of the complex muscle physiology. To obtain pharmacokinetic and toxicity data that can be predictive for human drug responses, we studied micro-injection of compounds into a previously developed human bio-artificial muscle BAM model 32 , 33 , This model system could bridge the gap between 2D cell culture and animal models, while immediately using human instead of animal tissue.
Injection of drugs in engineered tissues has not been described thus far and could be a valuable non-clinical model to assess compound toxicity, metabolism and release profile. BAMs are formed by mixing muscle cells, isolated from muscle biopsies, in a natural hydrogel in a silicone mold containing two attachment points.
Over a one week period, the cell-gel mix contracts to form multinucleated myofibers well-aligned in the direction of the attachment points.
Such a model can be used to study mechanisms underlying muscle development, skeletal muscle innervation and vascularization 33 , Furthermore this model can be used as an in vitro disease model for muscular disorders and drug screening. By using muscular dystrophic muscle cells in the BAM model, Vandenburgh et al.
Another example of the potential of the BAM model for drug screening purposes describes the positive effect of insulin-like growth factor 1 and the negative effect of a statin on the morphology and contractility of the human BAM. These effects are also observed in human muscle in vivo and show the predictive behavior of the BAM model for skeletal muscle drug screening While this BAM model has also been evaluated in ex vivo gene therapy studies 39 , this model has never been implemented in the context of IM injections.
The use of human cells in this 3D muscle model can improve the predictability of the human response on candidate drugs. This would increase the success rate of the compound in later clinical trials and at the same time reduce the cost of the drug discovery process 5. We show that several parameters such as compound toxicity, retention, release and metabolism of injected test compounds can be measured in this model system. The effect of myofiber composition in the BAM model on the release profile and metabolism was assessed as well.
Two compounds were chosen for injection in the BAMs with an in-house micro-injection system based on the ability to measure the amounts of both released parent compounds and their corresponding metabolites. The metabolic reactions that are quantified in the tests, namely hydrolysis and reduction, are two reactions that can occur during drug metabolism. We also determined compound toxicity on the BAMs by measuring creatine kinase release in the medium, which clearly increased with increasing toxic insult.
In short, we show that the BAM is an injectable human 3D cell culture model which can reflect compound toxicity and release from muscle-like tissue and is capable of metabolizing the injected compounds, a process that is also relevant to the in vivo situation.
A human skeletal muscle cell population was isolated from a biopsy from the vastus lateralis muscle of a year old Caucasian man 2 days post-mortem Human Body Donation programme of KU Leuven University, Prof.
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Vereecke as described in In the mixed muscle MM cell population, myoblast percentage and fusion index were determined as described in C2C12 mouse myoblast cell line was acquired from Sigma. Using sterile forceps and scalpel, excess connective tissue and fat was removed from the tissue sample.
Using 1. BAMs were engineered as previously described 33 , Briefly, C2C12 mouse myoblasts, human myoblasts M or human mixed muscle MM cells were trypsinized with 0. Medium was replaced every 2 days and BAMs were kept in culture for 7 days prior to microinjection. Next, HBSS was removed to perform the injection. The syringe with glass capillary could be exactly positioned using a 3D stereotactic system. Each BAM or strip was injected 1 to 4 times with 0. For visualization and optimization of the injection procedure, Trypan blue Thermo Fisher was used.
In case of multiple injections, different areas of the BAM were injected. All compounds in the washing steps were considered to be direct reflux. Effectively injected compound was calculated by subtracting the amount of refluxed compound from the total amount of compound that was recovered. Microinjection setup. The toxicity of an injected compound was determined by measuring creatine kinase CK release. Absolute concentration is proprietary so all concentrations are indicated as an x-times concentrated solution. All samples were added in duplicate and a 0. Immunocytochemistry was performed in order to characterize the myoblast M and mixed muscle cell MM population used in the injected BAMs.
The percentage of myoblasts and their fusion index was determined by a desmin and tropomyosin immunofluorescence staining. Next, cells were permeabilized in blocking buffer containing 0. The percentage of myoblasts in a muscle cell population was defined as the ratio of desmin positive cells to the total number of cells identified by the DAPI-stained nuclei. For each replicate — cells were counted. Fusion index was defined as the ratio of nuclei in tropomyosin positive cells to the total number of myoblast nuclei in the population.
Samples were stored in PBS in the dark until visualized. Myofiber alignment was determined by the standard deviation of the angles of the myofibers compared to the mean alignment direction. A lower number thus indicates better alignment. In human BAMs number of nuclei per fiber were counted on 2D images. All samples were run in duplicate. After each cycle, Sybr Green fluorescence was recorded. Number of replicates refers to number of analyzed images for microscopic analyses, to the number of wells containing 2D cells treated with a compound, to the number of biological samples for quantitative real-time PCR analysis or to the number of BAMs for thickness analyses and microinjections.
For comparing several normally distributed groups, a one-way ANOVA was used with a Bonferroni multiple comparison post test.
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