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Liquid biopsies are becoming an integral part of biomedical research and translation but are challenged by the fact that their markers are typically a vast minority in a mixed population in clinical samples such as blood. Therefore, the liquid biopsy marker must be purified/enriched prior to downstream analyses. There are several purification platforms for liquid biopsy markers including those that are specific for a particular sub-type (i.e., affinity selection) and those that select the biomarker irrespective of sub-type. For example, in the case of extracellular vesicles (EVs), isolation based on size will isolate all EVs within a certain size range but isolates both normal and diseased cells’ EVs. In the case of affinity selection, it will isolate EVs secreted from only diseased cells. In this presentation, I will discuss different microfluidic selection/purification strategies for EVs as a case example for the analysis of liquid biopsy markers from discovery (bench) to the clinic (bedside). I will then briefly discuss the use of microfluidics and EVs for two clinical applications: (i) the isolation of CD15 expressing EVs for the diagnosis of acute ischemic and hemorrhagic stroke using their mRNA expression analysis; and (ii) the early detection of ovarian cancer using EVs secreted from the Fallopian tubes of patients with the analysis of their miRNA content to improve test positivity. Due to the complexity of the analysis (i.e., enrichment followed by exosomal RNA analysis), I will also discuss the use of integrated and modular microfluidic systems for automating the workflow to facilitate testing in a clinical environment.

The small intestine is a dynamic tissue that continuously renews itself while performing essential functions such as nutrient absorption, barrier protection, and hormone secretion. My laboratory had developed an architecturally accurate 3D human small intestinal tissues with crypts-villi of the size and density in a living human. Cells of the crypts/villi are fully polarized with respect to luminal-basal orientation. Importantly the model displays a stem cell compartment in the crypt niche with cells migrating up the crypt/villus long axis, making fate decisions to place fully differentiated cells on the villus. Both living and apoptotic cells are extruded at the villus tips as the cells reach the end of their one-week lifespan. Importantly all cell types are present in their expected locations along the crypts/villi including Paneth cells in the stem cell niche, goblets cells throughout the crypts/villi and absorptive enterocytes localized to the villi. Enteroendocrine or hormone secreting cells of all types are present throughout the crypts/villi. When combined with a macroporous scaffold, stromal cells e.g., immune cells and fibroblasts can be included in the small intestine. Since these tissues are generated from patient-derived biopsies, they provide powerful tools for studying gastrointestinal disorders such as inflammatory bowel disease and malabsorption syndromes as well as drug discovery.

Interest in 3D printing for microfluidic fabrication remains high, but manufacturer-reported resolution still does not reliably predict the interconnected negative features that define device function. That outcome depends on pixel-scale irradiance, resin properties, process automation, and whether design software can express the controls the printer actually has.

We quantify this with pixel impulse response (PIR) measurements on Asiga DLP, Elegoo LCD, and Anycubic DLP-XPR printers as well as our custom printers, clarifying when commercial tools suffice and when custom optics and dose control are required. A family of nine resins is characterized for biocompatibility, Young’s modulus, Shore hardness, and solvent resistance so materials can be matched to valves, membranes, cell-facing structures, and assay chemistry.

These options are implemented in OS1, an open automated DLP 7.6 micron pixel platform (CERN-OHL-S) with automated planarization, focus correction, power control, and grayscale irradiance flattening, and in PyMFCAD ('pip install pymfcad') to design devices and write print files that expose the same controls. Examples include a device with 11,200 valves, uniform 7 µm isoporous membranes across the full build area, and stepped-height channels that extend colorimetric dynamic range by making optical path length a designed variable.

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Precision medicine is the paradigm to develop treatments for patients based on molecular-targets that are effective in vivo when administered. That is, one must not only be able to identify molecular and cellular targets that are the source of disease but also understand how these targets behave inside the body based on physiological principles. Recent developments in microfluidics have contributed to burgeoning precision medicine fields such as liquid biopsy, immunotherapy, single cell analysis, molecular , and microphysiological systems. Since microfluidics bridges the scales of molecular, cellular, tissue, and can even recapitulate organ and circulatory functions of the body it is the ideal platform technology to develop personalized medicine. Adoptive cell therapy (ACT) involves the processing of blood from a donor to isolate cells (e.g. T cells) for genetic manipulation followed by reinfusion back into patients. Specifically for CAR T cell therapy, genetic coding material (e.g. DNA, mRNA) is inserted into the T cells to express chimeric antigen receptors to target biomarkers of cancer cells and trigger an activated immune response towards the tumor of interest. This process that starts from blood drawn from one person and ends with specialized engineered cells delivered to the patient includes multiple tedious and costly steps, and can require a long time that the patient may not have. In this talk I will introduce three microfluidic platforms developed in my lab and applied to the process of cellular engineering and immunotherapy. First, a lateral cavity acoustic transducer (LCAT) was used for processing blood samples, isolating immune cells, transfecting cells, and finally expanding T cells to scale up for treatment. We developed the acoustic electric shear orbiting poration (AESOP) device to uniformly deliver genetic cargos into a large population of cells simultaneously for high quality transfected cells with controlled dosage delivery of different genetic cargos. Second, we constructed bottom-up artificial antigen presenting cells (aAPCs) for antigen-specific T cell activation based on droplet microfluidics. Finally, I will introduce a new droplet-based platform that is capable of studying the 3D cell morphology of both the cell surface and also its intracellular constituents to further understand immune cell activation status.

What are the areas that must be considered to carry a microfluidic or organ-on-a-chip (OoaC) idea from brilliant product concept to CAD layout, CFD model, prototype, beta and eventually launched product? How can you communicate your company’s capabilities in the language of your client? As an investor or start-up target, what are some key product and business areas to focus on for a due-diligence investigation — from either side of the table? I’ll illustrate how I support clients in all of these areas ... to help them hit their milestones before funds run out, the opportunity passes, or the competition steals their lunch!

Based on 13+ years of consulting to start-ups, SMEs and multinationals alike on a variety of technical product development, IP landscaping, and investment due diligence projects, a survey of these scope areas will be provided, along with an outline of how these projects typically proceed, for each project type.

Liquid biopsy disease biomarkers are enriched in extracellular vesicles (EVs). However, EV diagnostics face several daunting obstacles. They must have extraordinary sensitivity (~fM LOD) and selectivity from interference by far (by 3-6 logs) more abundant dispersed molecules and EVs from non-diseased cells. They also need to isolate specific EV subsets without pretreatment steps that lead to significant analyte loss. I will review an array of nanotechnologies from our lab that can circumvent these EV-specific issues and introduce our current effort to integrate these modules with a SPION immunobead technology with antibodies that can be cleaved by orthogonal techniques. Some of these technologies exploit the specific size range of EVs: a high-throughput conic-nanopore ultrafiltration technology for biomarker discovery (being commercialized by Aopia Bio, Cell Comm and Signaling, 2026; ACS Nano 2023), an interference-free Brownian rotation assay of EV-conjugated ImmunoJanus particles (Nature Biomedical Eng, 2026), a surface acoustic wave sensor to estimate the size of EVs/lipoproteins (Analytical Chem, 2026) and a magnetic nanoporous membrane for capturing SPION immunobeads (CommBio, 2022). Some are charge sensors to quantify EVs with mulitiple colocaized markers with minimum Debye screening and interference from weakly charged non-targets: membrane sensors for supermeres (Sci Rep, 2026), EVs(Comm Bio, 2024) and lipoproteins(Nature Comm, 2023) and isoelectric separation of different nanocarriers (ACS Nano, 2023). Some are activity assays to probe activities of EV surface enzymes (Biosensors and Bioelectronics, 2026). Others are optically based to allow single-EV resolution of colocalized proteins without cross-talk (Biosensors and Bioelectronics, 2025). Other than protein affinity and activity EV assays, the integrating magnetic beads also allow us to profile miRNA and mRNA from a subset of EVs (Small, 2022; Analytical Chem, 2021). We report some small clinical trials (for GBM, CRC, CVD, PDAC, AD) that demonstrate such integrated multiomic and multiplexed EV assays significantly elevate disease screening performance (with AUC>90% for all trials).

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Cardiovascular disease and underlying atherosclerosis is developing over decades in humans often without any symptoms until an event occurs. Most common events are myocardial infarction, stroke and increasingly periferal artery disease with walking pain, open wounds and amputations.

We are using plasma extracellular vesicle proteins to exclude atherosclerotic stenosis of the major arteries of the heart to reduce cost and burden on hospitals. Next to this, we use plasma extracellular vesicles proteins to identify the patient after a first cardiovascular event who has a high risk for a second cardiovascular event and needs add-on therapy or has a low risk and does not need a surgical intervention. Furthermore, we initiated a biobank of patient with an acute thrombotic occlusion in the major leg arteries to investigate using plasma extracellular vesicle proteins to determine which patient is at risk for a severe bleeding (eg hemoragic stroke) or which patient can safely undergo thrombolysis to remove the thrombus. In these studies, we seek differences in disease and in extracellular vesicle content are taken into account.

This presentation will highlight translational research from the UC Davis Center for Bioengineering in Medicine, where engineering principles are integrated with biology and clinical medicine to develop innovative biologic, cellular, nanomedicine, and genetic therapies across the lifespan. Using the CuRe Trial as a translational roadmap, Dr. Wang will describe the development of engineered placenta-derived mesenchymal stromal/stem cell technologies for fetal repair of myelomeningocele, from laboratory discovery and large-animal validation through pre-IND and IND-enabling studies, GMP manufacturing, regulatory interactions, and implementation of the world’s first FDA-authorized stem cell clinical trial for in utero treatment of spina bifida. Phase 1 safety results from this first-in-human study were recently published in The Lancet. Building on this translational foundation, the presentation will discuss emerging platforms that extend the concept of “engineering biology for cures,” including engineered stem cell-derived extracellular vesicles for tissue-targeted delivery, regenerative signaling, and repair, as well as lipid nanoparticle-based nonviral systems for in utero delivery of gene-editing cargos to treat monogenic diseases. Together, these programs illustrate a translational framework for moving engineered biological delivery systems from discovery toward clinical application.

The surface charge of lipid nanoparticles (LNPs) is set by the ionizable lipid and the surrounding pH, and zeta potential measured across pH reports the net particle charge, which has been linked to delivery efficiency and off-target expression. This presentation describes how the ZetaView® Evolution nanoparticle tracking analyzer measures size, concentration, zeta potential and fluorescence at the single-particle level for LNPs and extracellular vesicles (EVs). Concentration Scanning Technology increases the measurement volume from 33.3 nl to 220 nl and analyzes up to 20,000 particles in 30 seconds, making concentration measurement calibration-free. A sensitivity-improved CMOS camera detects particles carrying fewer than 20 AF488 molecules, and fluorescence NTA (F-NTA) with CD9, CD63 and CD81 detection kits quantifies labeled EV subpopulations. Zeta potential is measured in scatter and fluorescence mode to assess the colloidal stability of all particles or of specific subpopulations. Application examples with HEK- and MSC-derived EV preparations show quality control of size, concentration, purity and zeta potential, and monitoring of concentration and purity across depth filtration and tangential flow filtration.

Autosomal dominant polycystic kidney disease (ADPKD) is a prevalent genetic disorder leading to renal failure. Current treatments provide limited benefits and do not address the underlying genetic defect. Here, we develop targeted lipid nanoparticles (LNP) and extracellular vesicles (EVs) as platforms for the delivery of mRNA, microRNA, and protein to renal cells. A focused LNP library identified an optimized formulation, which, when functionalized with targeting peptides, enabled selective delivery to cyst-forming cells. In ADPKD mouse models, systemic administration of therapeutic LNPs restored protein expression, reduced cyst burden, and suppressed fibrotic and inflammatory signaling in ADPKD models. Additionally, EVs derived from urine from wildtype backgrounds contained cargo missing in the disease and inhibited disease progression in vivo. These findings demonstrate that LNPs and EVs can be used to restore renal function and reverse disease in genetic kidney diseases such as ADPKD.

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Extracellular vesicles (EVs) are promising therapeutic carriers secreted by living cells. However, conventional surface engineering of EVs is slow, noisy, and requires tedious cell culturing. Here, we present a cell-free platform that programs or mimics EVs. The approach synthesizes membrane and surface proteins in vitro and inserts them co-translationally into synthetic or native EVs. Our platform consisted of high-throughput and cell-free engineering of nanovesicles, machine learning, and high-resolution single-EV measurements. In addition, we study the efficacy of the synthetic EVs for treating various diseases, including neuroprotection and cell/tissue damage. Our cell-free platform enables the rapid prototyping of EVs, opening doors to creating superior EV-based therapeutics for broad biomedical applications.

Lipid nanoparticles (LNPs) have great potential for delivering nucleic acids, however they trigger the production of inflammatory cytokines and this limits their medical applications. Developing LNPs that do not trigger the production of cytokines is challenging because the LNP’s ionizable lipid and the process of endosomal disruption are the major sources of LNP toxicity, and are also essential for delivering nucleic acids. In this presentation, I will present a new class of LNPs that switch their charged states between the pHs of 7.4 and 4.0, and can deliver nucleic acids efficiently without triggering inflammation, termed the switchable nanoparticles (SNPs). The SNPs deliver mRNA and pDNA as efficiently as traditional LNPs in vitro and in vivo, but do not activate the TLR4, complement, galectin-8 and platlet activating factor (PAF) signaling pathways, and consequently have lower toxicity than traditional LNPs. In addition, SNPs are better at treating LPS-induced acute lung injury than traditional LNPs because they do not exacerbate pre-existing inflammation. We anticipate numerous applications of the SNPs because of their ability to deliver nucleic acids efficiently without triggering inflammation.

Univercells Technologies has developed the scale-X™ structured fixed-bed bioreactor platform. The spiral wound, bi-layer design of the scale-X fixed-bed provides a low shear environment for achieving high cell densities and high cell specific productivity. scale-X bioreactors are available from bench to commercial size (0.5m² to 600m² cell growth area) in a directly scalable format. These characteristics shorten time to market while de-risking scale-up and enabling significant cost savings. Originally designed for vaccine and viral vector manufacturing, the platform has also been successfully used for extracellular vesicles and stem cell manufacturing. This presentation will highlight the design of the scale-X bioreactor and its application to extracellular vesicle production in several case studies.

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30 years after the introduction of the concept of the “miniaturized total analysis systems (µTAS) and about 20 years after the gold digger’s frenzy about how this technology would revolutionize all aspects of chemical, biological or diagnostic applications, the technology has matured but is still a key driver to ease and improve analytics and diagnostics for a wide variety of applications.
The course will provide a broad overview of all aspects of microfluidics (lab-on-a-chip) technology as enabling technology for new product development strategies, manufacturing technologies, application cases, markets as well as aspects of commercialization and latest trends in research. Recent product examples will be presented as well as lessons learned during all stages of the development and commercialization process of microfluidic-enabled devices.

Furthermore, the second half of the course will be dedicated towards practical experience for the participants: With hands-on-tests first the basic operation of microfluidic standard modules will be introduced, focusing on fluidic interfaces, microfluidic modules like mixers, droplet generators, how standardization is continuously applied and how instrument modules and pumps complement the microfluidic devices.

Secondly, the way to plan and conduct product developments in a sped-up way by using and combining existing modules will be introduced with a set of microfluidic modules the participants can freely combine to simulate own experiments.

**Course Outline*
The course will be divided into a theoretical and practical part.

The theoretical part will cover:
1. Introduction into microfluidics
2. Physical principles
3. The reagent choice
4. The material choice
5. Development strategies
6. Case studies: Applications and products
7. The way towards commercialization
8. Lessons learned
9. Research trends
10. Conclusions

The practical part:
1. How to use microfluidic: The basics for a quick start with microfluidic modules, fluidic interfaces, handling frames, accessories, pumps and instrument modules.
2. How to plan a development start with microfluidic modules: For a given development task, the participants should choose from microfluidic modules to build up a test microfluidic system from single building block as microfluidics LEGO.

Learning Outcome:
1. An overview on microfluidics as technology and its benefits
2. Understanding microfluidics as a real system approach and the respective complexity: microfluidic chip, instrument, assay and reagents
3. Identifying the critical elements for the development of microfluidic systems
4. How to build a team to develop a microfluidic system
5. How to work with microfluidic modules and understanding the modular concept for system development with hands-on-experiments.
Target audience:
Researchers and technicians from academia and industry with interest in innovative technologies; technical sales and marketing personnel who want to get an introduction in the field. Technical and company management who need to evaluate technologies for strategic decisions.

Demonstrations:
The demonstrations will be done with various microfluidic modules showing mixing, droplet generation and combining microfluidic modules.

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Antigen-presenting cell-derived extracellular vesicles (APC-EVs) are essential immune mediators in cancer immunotherapy. They carry immunogenic factors, such as class I major histocompatibility complexes (pMHC-I), CD80, and CD86, which engage T cell receptors (TCRs) to form an immune synapse that could prime CD8+ T cell activity against tumors. Clinical trials using APC-EVs have shown that a lack of high-quality APC-EV isolates, a lack of advanced molecular engineering methods to engineer pMHC-I, and varied patient MHC-I/TCR repertoires have impeded their translation as an effective cancer immunotherapeutic. To address this translational challenge, a foundational molecular engineering platform was developed to design APC-EVs with peptides that facilitate high-quality immune synapses and prime CD8+ T cells to initiate TCR-restricted killing of tumors. This presentation will introduce our recently developed peptide engineering approach for rapid, large-scale generation of high-quality APC-EVs employed in immunogenic screening across various patient immune profiles and facilitating the development of precision cancer immunotherapies.

Demeter EVPrep is a novel EV-isolation tool that separates EVs using dielectrophoresis and semiconductor technology. The Demeter EVPrep system provides a fully automated, hands-off method for obtaining high quality EV preps from various sample types. Early adopters of this technology observe improved yield and purity of EV isolations, as well as compatibility with downstream analyses like mass spectrometry.

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The extracellular matrix plays a pivotal role in wound repair, cancer, aging, disease, and more, and recently, a new type of extracellular vesicle (EV) was found to reside within this matrix. These extracellular vesicles, termed matrix-bound nanovesicles (MBVs), are distinct from other types of EVs and recapitulate many of the features of the native extracellular matrix from which they are derived. I will present the recent work ongoing in my lab on the characterization and engineering applications of MBVs. Specifically, we characterize differences of MBVs from other types of EVs, including their therapeutic potential as drivers of wound repair and their link to the extracellular matrix. I will also discuss our methods of combining MBV with biomaterials to control their delivery to overcome one of the major bottlenecks in EV therapeutics: rapid systemic clearance by the body.

Tumor extracellular vesicles (EVs) have demonstrated promise to serve as therapeutic cancer vaccines for treating solid tumors, but are limited by the modest cytotoxic T lymphocyte responses and therapeutic benefit. In this talk, I will share our recent work on metabolic tagging and targeting of tumor EVs and further development of robust tumor EV vaccines.

Exosomes are naturally cell-derived nanoscale extracellular vesicles with exceptional potential for targeted drug delivery due to their biocompatibility, low immunogenicity, and ability to cross biological barriers. This presentation will highlight our laboratory’s recent advances in the genetic engineering of exosomes for precision medicine applications. Topics will include the development of programmable membrane scaffolds for cargo loading, molecular targeting, surface display, and real-time imaging of engineered exosomes in living human cells. Recent findings demonstrating how membrane scaffold architecture influences exosome biogenesis, intracellular trafficking, cargo incorporation, and therapeutic function will be discussed. Applications in inflammation, lysosomal storage disorders, Alzheimer’s disease, and targeted cancer therapy will be presented, along with emerging strategies exploiting diverse viral and cellular membrane scaffolds to improve therapeutic specificity, efficacy, and translational potential.

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This presentation will demonstrate the dual application of extracellular vesicles (EVs); first, as disease biomarkers, and second, owing to their natural uptake by cells, as drug delivery vehicles that can carry therapeutic payloads to cells and tissue. We will report on a microfluidic chip for efficient affinity isolation and preconcentration of EVs. Microfluidic chip can also serve as a solid-phase platform for EV modification and EVs’ surface bioengineering. Chips are made from thermoplastics and are amenable to high-volume production, thus supporting the testing and clinical validation of these biomarkers. Following selection or bioengineering, EVs are released from the surface for enumeration, visualization, molecular profiling of their cargo, or treatment of model cell lines. By analyzing mRNA derived from phenotypically distinct EVs isolated from plasma of breast cancer patients we secured information on molecular subtypes of cancer cells, which can guide targeted therapy. Finally, we will show how breast cancer-derived EVs can reprogram recipient cells, altering cells phenotype at the mRNA level.

The development of Point of Care (POC) devices for the early detection of cancer present both technical and practical problems, including cost, need for a venous blood draw, complex assay procedures, and impracticality of doing time course measurements. Blood Spot Cards (BSC) offer a gateway to POC devices as they provide a fast, efficient, and cost-effective method for collecting patient blood samples. However, the small sample size ~5ul-10ul often contains limited amounts of cancer-related cf-DNA and exosome biomarkers. To overcome these problems new assay methods, utilize fluorescent charge-changing peptide (CCP) substrates for the detection of cancer-related protease biomarkers Trypsin, Chymotrypsin, Cathepsin-S and MMP-2. In the case of Pancreatic Ductal Adenocarcinoma (PDAC), elevated levels of these biomarkers could be detected in <1ul of patient blood and plasma samples, including in BSC samples stored for more than 10 months. New POC devices based on the CCP assay procedures together with BSC samples offer a promising diagnostic technology for the early detection of cancer and disease time course monitoring.

Angelman syndrome (AS) is a rare neurodevelopmental disorder caused by deficiency of neuronal UBE3A expression. Although intracellular consequences of UBE3A deficiency have been extensively studied, its effects on extracellular communication remain less understood. This talk examines how altered extracellular vesicle (EV) release and function may contribute to synaptic and cognitive deficits in AS model mice. We investigated EV release from synaptosomes of brains from wild-type (WT) and AS mice and the effects of WT neuron-derived EVs on neuronal structure and behavior. Synaptosomes from AS mice exhibited reduced EV release following depolarization or activation of the lysosomal calcium channel TRPML1. These findings implicate disrupted activity-dependent and endolysosomal regulation of EV secretion. Proteomic analyses further revealed alterations in EV-associated proteins related to synaptic function, mitochondrial processes, and protein homeostasis. Treatment with WT neuron-derived EVs improved dendritic spine density and morphology in AS neurons. Systemic administration WT synaptosome-derived EVs also improved learning and neuronal structural measures in AS mice.

Together, these findings support the contribution of impaired EV-mediated communication to AS pathology and identify restoration of physiological EV signaling as a potential therapeutic strategy. They also highlight unresolved questions concerning the active EV components, recipient-cell responses, and the relationship between intracellular UBE3A function and EV-mediated effects. Understanding these mechanisms may reveal new approaches to restoring synaptic function in neurodevelopmental disorders.

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Tobacco use remains a leading cause of preventable death worldwide, and nicotine's addictive properties are driven largely by its actions on the mesolimbic dopamine system, including dopaminergic projections from the ventral tegmental area (VTA) to the medial prefrontal cortex (mPFC) and nucleus accumbens (NAc). Extracellular vesicles (EVs) are emerging as important mediators of intercellular communication in the brain, yet how drugs of abuse affect EV signaling remains largely unexplored. Using transgenic mice generated by crossing a CD81-NeonGreen reporter line with DAT-Cre mice, we combined super-resolution and hyperspectral imaging to characterize dopaminergic neuron-derived EVs in the cerebrospinal fluid (CSF), mPFC and NAc following acute nicotine exposure. Nicotine increased EV release in both the mPFC and NAc and altered vesicle biodistribution in male and female mice. These findings identify EV signaling as a previously unrecognized target of nicotine within the reward circuit and suggest that EVs may be clinically relevant in nicotine dependence.

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Lipid nanoparticles are the most clinically advanced nonviral gene therapy platform. A key to unlocking their potential is achieving tropism in organs beyond the liver. In this presentation, I will summarize our work on modifying lipid nanoparticles with peptide ligands to reprogram their tropism to cell types outside the liver.

BioCapZ™ is a programmable protein nanocarrier designed to overcome persistent limitations in RNA and biologic delivery, including poor tissue
specificity, invasive administration, formulation complexity, and storage constraints. A calcium-responsive disassembly-reassembly mechanism enables mild, aqueous loading of mRNA, plasmid DNA, proteins, small molecules, and CRISPR-Cas9 ribonucleoproteins. The exterior surface can be modified with targeting ligands, nanobodies, antigens, or imaging agents, supporting applications in precision delivery, mucosal vaccination, and theranostics. Researchers are developing its stable protein shell for oral and intranasal administration and scalable, fermentation-based manufacturing. Current translational directions include targeted cancer therapy and imaging, universal mucosal vaccines, and non-invasive gene-editing delivery for gastrointestinal disorders. BioCapZ therefore represents a unified platform that connects payload protection, tissue targeting, controlled delivery, and manufacturability across multiple therapeutic modalities.

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