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Preclinical models of IBD
Introduction • IBD is a group of chronic relapsing inflammatory conditions of the colon & small intestine • It is chronic gastrointestinal disorder characterized by intestinal inflammation and mucosal tissue damage initiated and perpetuated by a dysregulated immune response along with several intra- and extra intestinal manifestations, including autoimmune phenomena
• 2 major manifestations of IBD are  Ulcerative colitis  Crohn’s disease  https://www.youtube.com/watch?v=iefghc2g91M
signs & symptoms • Diarrhea • Rectal bleeding • Passage of mucus • Abdominal pain • Anorexia • Fever • Weight loss Mechanisms of inflammatory bowel disease.
• Mechanisms of inflammatory bowel disease. (FOOTNOTE OF FIGURE) • The intestine shelters a large diversity of microbiota that are in perfect balance (symbiosis); • balance can be affected by many factors leading to the appearance of pathogenic bacteria that can alter the intestinal barrier and lead to the development of inflammatory bowel disease. • The stimulation of the mucosal immune system may occur as a result of the penetration of bacterial products through the mucosal barrier, leading to their direct interaction with immune cells, especially dendritic cells and lymphocyte populations, to promote a classic adaptive immune response. • Alternatively, bacterial products may stimulate the surface epithelium, possibly through receptors that are components of the innate immune-response system; the epithelium can, in turn, produce cytokines and chemokines that recruit and activate mucosal immune cells. • Activation of classic antigen-presenting cells, such as dendritic cells, or direct stimulation through pattern-recognition receptors promotes the differentiation of type 1 helper T-cells (Th1) in patients with Crohn's disease (shown here) or, possibly, atypical type 2 helper T-cells in patients with ulcerative colitis. The stereotypical products of Th1 promote a self- sustaining cycle of activation with macrophages. In addition to producing the key cytokines that stimulate Th1 (interleukin-12, interleukin-18, and macrophage migration inhibitor factor), macrophages produce a mix of inflammatory cytokines, including interleukin-1, interleukin-6, and most notably tumor necrosis factor, which target a broad variety of other types of cells. The latter include endothelial cells, which then facilitate the recruitment of leukocytes to the mucosa from the vascular space. Most important, these functions may be altered either by genetically determined variants, as exemplified by germ-line mutations in the gene encoding NOD2, the product of the IBD1 locus, in some patients with Crohn's disease, or by environmental factors.
Screening Models Sodium trimethylsilylpropanesulfonate (DSS) Sodium trimethylsilyl propionate (TSP)
https://blog.crownbio.com/mouse-models-of-inflamatory-bowel-disease
Chemically induced colitis 1. Oxazolone induced • Oxzolone is haptenizing agent (To react an antigenic compound) • widely used to induce colitis in mice in order to evaluate the pathological processes involved in the perpetuation of ulcerative colitis. • This model of colitis generates an immune response mediated by Th2 cells • resembles human ulcerative colitis on the basis of manifestations of inflammation of the mucous membranes, epithelial microulcerations, and histopathological changes in the distal colon
• Procedure : Enema (rectal administration) of oxazolone (3%) with ethanol would induce colitis it cause colitis earlier than other other chemicals Effects :Initial symptoms starts second day after the enema, and symptoms diminished after 10–12 days. Colitis, accompanied by ulcers, was localized in the distal colon.
L-10 Knockout Model for IBD Interleukin-10 (IL-10) is a critical anti-inflammatory cytokine that regulates immune responses in the gut. Mice genetically engineered to lack IL-10 (IL-10⁻/⁻) spontaneously develop chronic enterocolitis resembling human IBD, particularly Crohn’s disease.
Generation of the IL-10 Knockout Mouse •Strain: C57BL/6 or BALB/c background. •Genetic Engineering: Targeted deletion of the Il10 gene via homologous recombination in embryonic stem cells. •Breeding: Heterozygotes are bred to produce homozygous IL-10⁻/⁻ mice. Disease Manifestation •Spontaneous colitis appears typically after 3–8 weeks of age. •The severity may depend on: • Genetic background (e.g., C57BL/6 mice develop milder disease). • Microbiota composition (germ-free IL-10 KO mice do not develop colitis).
Feature Description Histopathology Mucosal thickening, lymphocytic infiltration, crypt abscesses, ulcerations Cytokine profile Increased TNF-α, IL-1β, IFN-γ Immune response Enhanced Th1 and Th17 responses Microbiota dependency Colitis is driven by commensal bacteria; germ- free mice remain disease- free Features of IBD in IL-10 KO Mice
Applications in Research •Studying pathogenesis of IBD. •Testing anti-inflammatory therapies (e.g., biologics, probiotics, dietary interventions). •Exploring the gut microbiome–immune system interaction. •Evaluating nutritional and environmental modifiers of IBD. Example Experimental Design
Group Treatment Purpose WT control No treatment Baseline comparison IL-10 KO No treatment Disease model IL-10 KO + Drug A Anti-inflammatory compound Therapeutic effect IL-10 KO + Probiotic Specific probiotic strain Microbiome-based intervention Study Plan
Ethical Considerations •Mice develop chronic inflammation and may require early endpoints or humane euthanasia. •Ensure IACUC approval or equivalent ethical clearance.
Limitations of the IL-10 Knockout Model for IBD 1. Microbiota Dependency •Colitis does not develop in germ-free conditions; the disease requires colonization with commensal gut microbiota. •Disease severity and progression vary based on microbiota composition, making reproducibility across labs difficult. 2. Lack of Human IBD Complexity •IL-10 KO mice primarily exhibit Th1-mediated inflammation, which closely models Crohn’s disease, but not ulcerative colitis. •Human IBD is polygenic and influenced by multiple environmental and immune factors; IL-10 KO mice lack this multifactorial complexity. 3. Chronic, Non-Relapsing Disease •Unlike human IBD, which is characterized by relapsing-remitting cycles, the IL-10 KO model shows progressive, chronic inflammation without natural resolution phases.
4. Systemic Effects of IL-10 Deficiency •IL-10 has widespread anti-inflammatory roles beyond the gut; IL-10 KO mice may develop extraintestinal inflammation (e.g., arthritis, prostatitis), complicating interpretation of gut-specific effects. 5. Genetic Background Sensitivity •Disease severity is strain-dependent (e.g., more severe in BALB/c than C57BL/6), which can lead to inconsistent results unless carefully controlled. 6. Not Suitable for Late-Onset IBD •Inflammation typically appears at a young age (3–8 weeks), which doesn’t mimic adult-onset IBD seen in many human cases. 7. Non-targeted Immune Deficiency •Complete IL-10 knockout affects multiple immune cells (T cells, macrophages, dendritic cells), making it hard to isolate the effects of individual immune pathways
Models of Chronic Kidney Disease (CKD)
Introduction to Chronic Kidney Disease (CKD) • - Definition and staging (GFR < 60 mL/min/1.73 m² for >3 months) • - Pathophysiology: Nephron loss, fibrosis, inflammation • - Clinical relevance: Progression to End Stage Renal Disease
Importance of CKD Models • - Study of renal mechanisms and pathophysiology • - Evaluation of new therapies and biomarkers • - Modeling comorbidities (e.g., diabetes, hypertension)
In Vivo Animal Models: Surgical • - 5/6 Nephrectomy: Simulates chronic renal failure • - Unilateral Ureteral Obstruction (UUO): Fibrosis model https://www.youtube.com/watch?v=CuT4kMW1c2c https://www.youtube.com/watch?v=z4MjyzlQc5Y
In Vivo Models: Chemical & Genetic • - Adenine diet: Tubular injury and fibrosis • - Cisplatin/Aristolochic acid: Nephrotoxic injury • - Col4a3 KO (Alport), db/db (diabetic nephropathy)
In Vivo Models: Hypertension-Induced • - Spontaneously Hypertensive Rats (SHR) • - DOCA-salt model: Hypertension and fibrosis
In Vitro CKD Models • - Cell lines: HK-2, podocytes, mesangial cells • - Organoids: 3D nephron-like structures from stem cells • - Kidney-on-a-chip: Real-time, flow-based analysis Podocytes are highly specialized epithelial cells that cover the outside of the glomerular capillary. Mesangial cells are contractile cells which pack the glomerulus and have features of smooth muscle. HK-2 cells are derived from a normal, human adult male kidney. The cell line has applications in toxicology research.
Model Selection Criteria • - Based on research goals: Fibrosis, inflammation, filtration defects • - Ethical/logistical considerations • - Relevance to human CKD and co-morbidities
Limitations of CKD Models • - Species differences and lack of chronicity • - Often single-factor models • - Ethical concerns and cost
Emerging and Advanced CKD Models • - Humanized mice and iPSC-derived organoids • - CRISPR/Cas9 gene-edited models • - AI-based simulation and in silico predictions
Conclusion • - No single model is ideal—combine in vivo, in vitro, and in silico • - Model choice depends on the hypothesis and translational goals • - Future lies in personalized and human-relevant systems
Animal Models of Diabetes Mellitus
Introduction • Diabetes Mellitus is a chronic metabolic disorder with two major types: • Type 1: Autoimmune β-cell destruction → Insulin deficiency • Type 2: Insulin resistance with progressive β-cell dysfunction • Animal models help in pathogenesis study, drug testing, and mechanism elucidation
Ideal Model Characteristics • Reproduces key features of human diabetes • Predictive for therapeutic efficacy • Genetically manipulable and cost-effective • Reproducible with measurable biomarkers
1. Surgical induced 5/7/2025 ● Partial pancreatectomy: usually anterior lobe is left intact Disadvantages ● Loss of α cells (glucagon and somatostatin loss) ● Loss of pancreatic enzymes (creates digestive problems) ● Difficult to achieve
5/7/2025 • naturally occurring chemical • toxic to the insulin-producing beta cells of the pancreas • chemotherapeutic drug for treating certain cancers of the islets of Langerhans • used in medical research to produce an animal model for type 1 diabetes. • It is also an antibiotic effective against Gram-negative bacteria. Chemically induced Type 1 Diabetes Models- STZ
5/7/2025 Mechanism ● Cell methylation ● Free radical generation ● Nitric oxide production
5/7/2025 Procedure Male Wistar Rat- 150-200 gm Group- Test, Standard, Control STZ. Dose- 60 mg/kg iv or ip ● Done in mice (175-200 mg/kg) and dogs (15 mg/kg for 3 days)
14 5/7/2025 Evaluation ● Blood glucose level is estimated. ● Compare the result of test with standard and control.
5/7/2025 ADVANTAGE • Selectivity towards β cells • Lower mortality rate • Longer diabetes induction Limitations • Rabbits and guniea pigs are resistant to its diabetogenic effect
Type 1 Diabetes Models- NOD Mouse Non- obese diabeties • Spontaneous autoimmune β-cell destruction • Strong MHC II association (H2-g7) • Mimics human T1DM immunology • Limitation: no ketoacidosis
Genetically induced Type 2 Diabetes Models- ob/ob & db/db • ob/ob: Leptin deficiency → Obesity, insulin resistance • db/db: Leptin receptor defect → T2DM progression • Used for obesity- • Leptin is a hormone your body releases that helps it maintain your normal weight on a long-term basis. The level of leptin in your blood is directly related to how much body fat you have. Leptin resistance causes you to feel hungry and eat more even though your body has enough fat stores. linked T2DM studies
17 5/7/2025 Principle ● Encephalomyocarditis virus (EMC-D) selectively infects and destroys pancreatic β-cell. ● Associated with chronic islet cell inflammation, elevation of islet cell antibody, and prolonged presence of viral RNA in the pancreas. ● Others: RNA picornavirus, Coxsackie virus Viral induced
Viral induced https://www.sciencedirect.com/science/article/pii/S0168822720308949
T2DM- Diet & Transgenic Models • High-fat diet induces metabolic syndrome and T2DM • HFD + STZ: Mimics human late-stage diabetes • Transgenic KOs: IRS1/2, GLUT4, PPARγ, RIP-DTR
Limitations & Summary • No model captures all aspects of human diabetes • Rodent models lack chronic complications • Species-specific immune and metabolic differences • Use combined models for better translational relevance
Animal Models in Cardiovascular Disease (CVD) Research
Introduction • Cardiovascular diseases (CVDs) are the leading cause of death globally. • Animal models are essential for understanding disease mechanisms and testing therapies. • CVD research includes studies on atherosclerosis, hypertension, heart failure, and arrhythmias.
Criteria for an Ideal Animal Model • Mimics human pathology closely • Genetic and physiological relevance • Reproducibility and reliability • Ethical acceptability and cost-efficiency
Small Animal Models – Mice • Transgenic and knockout models (e.g., ApoE-/-, LDLR-/-) • CRISPR/Cas9 for precise genetic editing • Advantages: genetic tools, cost-effective, short lifespan • Limitations: metabolic and size differences from humans
Rats in CVD Research • Models include Spontaneously Hypertensive Rat (SHR), Dahl salt- sensitive rat • Used for hypertension, heart failure, and vascular studies • Better physiological match for cardiovascular studies than mice
Large Animal Models – Pigs • Close anatomical and physiological similarity to humans • Used in studies of coronary artery disease and ischemia-reperfusion • Ideal for testing surgical techniques and medical devices
Canine Models • Historically used in arrhythmia and myocardial infarction research • Similar cardiac electrophysiology to humans • Use limited due to ethical concerns
Non-Human Primates (NHPs) • Genetically and physiologically closest to humans • Used in advanced drug and gene therapy research • Expensive and subject to strict ethical regulations
Induction Techniques in CVD Models • Surgical: coronary artery ligation, aortic banding • Dietary: high-fat diets in ApoE-/- mice • Chemical: isoproterenol, doxorubicin-induced models • Genetic: targeted mutations to study specific pathways
Applications of CVD Animal Models • Study of disease mechanisms like plaque formation • Drug development and safety assessment • Evaluation of new therapies (gene, cell, regenerative)
Limitations and Challenges • Species-specific responses limit translational accuracy • Ethical concerns with certain models • High cost and technical demands of large animals
Future Directions • Development of humanized mouse models • Use of iPSC-derived cardiac tissues for in vitro research • Integration of omics data and AI for predictive modeling
Summary • Animal models are vital for CVD research. • Each model has unique strengths and limitations. • Future efforts focus on ethical, accurate, and translationally relevant models.
Lecture 16 MICROBES OF MEDICAL SIGNIFICANCE BIOFILMS 6 2
Bacterial Biofilms: Introduction • In recent time, microorganisms have been discovered to evolved various mechanisms of surviving a harsh environment; among which are production of biochemical compounds such as biofilm, biosurfactants, and enzymes. • Biofilms are self synthesized extrapolymeric slimy matrix that encloses a sessile microbial community whose cells are characterized by their attachment to either biotic or abiotic surfaces (Vasudevan, 2014). • Biofilm consist of either a single or multiple species of microorganisms (Flemming and Wingender, 2010). • Biofilms promote and regulate metabolic activities, Provision of nutrients and protection (Vasudevan, 2014). 6 3 https://study.com/academy/lesson/what-are-biofilms-definition- formation-examples.html
6 4 Composition of Biofilm • Microbial cells • Extra polymeric substances, EPS (Oliveira and Cunha,2008) EPS provides essential nutrients, protection, architectural integrity, enables genetic and intracellular transfer (Donlan, 2000). Table 1: Composition of biofilm (Vasudevan, 2014). Do not memorize the matrix percentages S/NO Component Percentage of Matrix 1 Water Up to 97% 2 Microbial cells 2-5% 3 polysaccharides 1-2% 4 Proteins <1-2% (including enzymes) 5 DNA and RNA <1-2% 6 Ions Bound and free
Biofilm formation  Biofilm formation begins when microorganisms response to factors such as • Cellular recognition of attachment site, • Nutritional cues • Exposure of planktonic cells to sub-inhibitory concentration of antibiotics (Lieve van et al., 2012)  Stages in biofilm formation include, Adhesion/Attachment, adhesion of bacteria to a surface is a two step process; primary bacterial adhesion and secondary bacterial adhesion (Chelius and Duffy, 2013) Intracellular aggregation, formation of monolayer involving polysaccharide adhesin (PIA) similar to poly–N- acetylglucosamine (Helimann, 2011) Biofilm maturation, biofilm become complex, cells actively proliferating leading to increase in population density, Forms glycocalyx and mushroom- like structure (Chelius and Duffy, 2013; Niels et al., 2011). Biofilm dispersal, nutrients and Oxygen become depleted and biofilm begin to degrade and cells released from the mushroom like structure (Karatan and Watnick 2009; Hong et al. 2010; Rowe et al. 2010) 5
Fig1 Stages in biofilm formation. Adopted from Kirk E. Anderson Genetics and Microbial Ecology 66
Quorum sensing and Biofilm formation 67 https://www.youtube.com/watch?v=mQ43fuJJW7M&t=1s
Clinicial significance of biofilms 68 Bacterial Biofilms Antimicrobial tolerance Medical device related infections Chronic infections Modulation of immune response https://www.youtube.com/watch?v=aXb4Bg6DXE4 https://www.youtube.com/watch?v=0DSA_8t4-UA&t=48s
Biofilm and antimicrobial resistance. • Agents may form complex with EPS (Romling and Balsallobre, 2012) • Poor penetration of biocide agent resulting from EPS (Manavathu and Vazquez, 2014). • Passage of genetic determinant via horizontal gene transfer (Juhas, 2013). • Expression of molecular efflux pump (Bueno, 2014) https://www.youtube.com/watch?v=8VZH4GZLWZc
Medical device related infections 70 • Most medical implants such as intravascular catheters, Devices use in orthopedic shunts, Stent, suture are susceptible to biofilm formation (Romling and Balsalobre, 2012). • Provide a reservoir for recurrent and at times life threatening bloodstream and urinary tract infections (Desai et al., 2014). • Biofilm causes device failure
Biofilms and chronic diseases  Chronicity in diseases such as diabetic chronic wounds, • Cystic fibrosis, • Bronchopneumonia, • Otitis media, • Osteomyelitis (Bjarnsholt et al., 2009; Homoe et al., 2009; Tacconelli et al., 2009)  Biofilm triggers proinflammatory responses harmful to host tissues.  Responsible for delayed healing in diabetic wound (Zhao et al., 2013). 12 https://www.youtube.com/watch?v=dws3bEj2Cjg
Modulation of immune response EPS component(s) at times triggers proinflammatory response that fails to recognize host tissue as ‘self’ with detrimental effects (Nibali et al., 2014). Microbial biofilm mediated inflammatory response halts the chronic wound healing process at the inflammatory stage and prevent proliferation and healing (Manavathu and Vazquez, 2014). Lipopolysaccharides released from dental plaque initiate imflammatory immune response which triggers the release of inflammatory mediators (Benakanakere and Kinane, 2012). Bacteria-in-wound protected by biofilm from the host immune system. 13
Future direction for combating biofilm menace • Development of antibiofilm antimicrobial agents directed to targeting one or more of the major stages of biofilm development. • Extra polymeric matrix busting’ antibiofilm drug should have the ability to disrupt existing biofilm. • The use of bacterial/fungal adhesion resistant material for the construction of medical devices associated with biofilm. • Development of vaccines directed to targeting quorum sensing. 14
7 4 Lecture 17 Synthetic Biology For Clinical Research
 Synthetic Biology • Designing/redesigning new biological parts, devices and systems that do not exist in the natural world. 7 5 • The term appeared in the literature in 1980 to describe bacteria that had been genetically engineered using recombinant DNA technology. • Synthetic biology = Bioengineering. • The final goal is to be able to design biological systems in the same way engineers design electronic or mechanical systems.
  Potential applications of synthetic biology range very widely across scientific and engineering disciplines, from medicine to energy generation. For example, designed microorganisms might be capable of producing pharmaceutical compounds that are extremely challenging for existing methods of chemical or biological synthesis. What can synthetic biology achieve? 76 https://www.youtube.com/watch?v=rD5uNAMbDaQ
 • Bacteria or viruses could be programmed to identify malignant cancer cells and deliver therapeutic agents (Serrano 2007). • Viruses have also been engineered to interact with HIV-infected cells, which could prevent the development of AIDS (De Vriend 2006). Medical Applications • Construction of an artificial metabolic pathway in E. coli and yeast to produce a precursor (arteminisin) for an antimalarial drug (Martin et al. 2003, Ro et al. 2006).
• Biofuel: There are many ways of engineering microorganisms to produce carbon-neutral (or more environmentally friendly) sources of energy. For example, bacteria could be engineered to synthesize hydrogen or ethanol by degrading cellulose, although further work is needed to overcome technical barriers. 78 • Synthetic vaccines. The fact that synthetic biology can ‗start from scratch‘ means that new synthetic vaccines could be produced in response to viruses that themselves evolve rapidly, such as those that cause severe acute respiratory syndrome (SARS) and hepatitis C (Garfinkel et al. 2007).
  There are two factors which make the risk governance of synthetic biology potentially problematic. • The first is that synthetic biology (like genetic engineering) involves the production of living organisms, which by definition are self- propagating. • The second is that with the growth of the Internet and the routinisation of many biotechnological procedures, the tools for doing synthetic biology are readily accessible (Garfinkel et al. 2007). Risks related to synthetic biology 79
Ethical Issues – A certain Concern  Statements to the effect that the next 50 years of DNA evolution will take place ―not in Nature but in the laboratory and clinic‖ (Benner 2004:785), accompanied by inventions such as plants that produce spider silk, clearly challenge everyday understandings of nature and our place in it. 39 • Additionally, the flexibility of synthetic biology means that microorganisms could be created which are radically different from existing ones, and these microorganisms might have unpredictable and emergent properties (Tucker and Zilinskas 2006), making the risks of accidental release very difficult to assess in advance (De Vriend 2006).
Cell culture as Models system
Cell culture  Cell culture is the process by which prokaryotic, eukaryotic or plant cells are grown under controlled conditions. But in practice it refers to the culturing of cells derived from animal cells.  Cell culture was first successfully undertaken by Ross Harrison in 1907  Roux in 1885 for the first time maintained embryonic chick cells in a cell culture.
Why is cell culture used for? Areas where cell culture technology is currently playing a major role. Model systems for Studying basic cell biology, interactions between disease causing agents and cells, effects of drugs on cells, process and triggering of aging & nutritional studies Toxicity testing Study the effects of new drugs Cancer research Study the function of various chemicals, virus & radiation to convert normal cultured cells to cancerous cells
Types of cell culture • Animal cell cultures can be divided into two distinct groups depending on the number of cell divisions occurring during the process; 1. Primary cell culture •Primary cell culture is the first culture obtained directly from animal tissue via mechanical and chemical disintegration or enzymatic methods. •The cells of the primary cell culture are slow-growing cells that carry all the characteristics of the original tissue or cells. •Primary cell cultures can be subcultured to obtained other cultures that either continue to grow indefinitely or die after a few subcultures.
Types of Animal cell culture • 1. Primary cell culture
• Primary cell cultures can be further divided into two groups depending on the kind of cells present in the culture; • a. Anchorage-dependent/Adherent cells • The cells in the culture require a stable biologically inert surface for adherence and growth. • The surface should be solid and nontoxic as these cells are difficult to grow as cell suspensions. • These cells are usually obtained from the tissues of organs where the cells remain immobilized within the connective tissue. • Examples of adherent cells include kidney cells and mouse fibroblast STO cells.
• b. Anchorage-independent/ Suspension cells • These cells can grow efficiently as cell suspensions and do not require a solid surface for attachment. • These can be grown on liquid media continuously to obtain fresh subcultures. • The ability of the cells to grow as suspension depends on the source of cells as cells that remain as suspensions in the body are effective suspension cells. • Examples of suspension cells include blood cells that are vascular and remain suspended in the plasma.
• 2. Secondary cell culture • Secondary cell cultures are obtained after the primary cell cultures are subsequently subcultured over a period of time in fresh culture media. • The cells of the secondary cell cultures are long-lasting as these have a higher lifespan due to the availability of appropriate nutrients at regular intervals of time. • Secondary cell cultures are favored over primary cell cultures as these are more readily available and are easy to grow and preserve. • The cells of the secondary cell culture might not resemble that on the parental tissue as mutations, and genetic alterations might be introduced during the subculture process.
• 3. Cell Lines • A cell line is a group of cells that are formed from the subculture of primary culture consisting of a pure culture of cells. Cell lines usually display functional features that are close to the primary cells, but the genotype and phenotype of the cells can be modified. A cell line consists of several cell lineages with similar or different phenotypes. • Cell lines can be further divided into two groups based on the growth patterns of the cells;
a. Finite cell lines • Finite cell lines are cell lines where the cells in the culture divide for a limited number of times, after which they eventually die. • The cells in the finite cell lines can divide from 20 to 100 times before they eventually die and cannot divide anymore. • The number of cell division and lifespan depends on a number of factors like cell lineage differences, species, culture conditions, and media. • The cells of the finite cell lines grow as adherent cells on solid surfaces.
• b. Continuous cell lines • Continuous cell lines are cells that exhibit indefinite growth via subsequent subcultures. • The cells in the continuous cell lines grow faster to form an independent culture. The cells are immortal and can divide indefinitely. • The cells in the continuous cell lines can be transformed via genetic alterations and are also tumorigenic. • The transformed cells are formed from the normal primary cell cultures after treatment with chemical carcinogens or by infection with oncogenic viruses. • The cells are capable of growing to prepare higher cell density and can grow as suspensions on liquid media. • These cells can even grow on top of each other to form multilayered structures on the culture vessels.
• Examples of common Cell Lines • The following are some of the common examples of cell lines; • a. HeLa cell line • HeLa cells are one of the first continuous culture human cell lines with the help of cells of the cervical carcinoma. • These cells are used for processes like virus cultivation and preclinical drug evaluation. • b. HL 60 (Leukemia) • c. MCF-7 (breast cancer cells)
Confluency • Confluence （ Confluent monolayer ） is when the adherent cells cover the adherent surface of the culture vessel. When culturing adherent cells, the rate of cell growth can be determined based on the ratio of the cultured cells covering the adhesion surface of the culture vessel (cell occupied area ratio or confluency).
• Procedure or Protocol of Animal cell culture • 1. Growth Conditions • Animal cell culture requires the use of specific culture media that are more complex and specific than the basic culture media used for microbial growth. • Some of the important basic components of the media are inorganic salts, nitrogen source, energy source, vitamins, fat and fat-soluble vitamins, growth factors, and hormones. In some cases, pH buffering systems and antibiotics are also added. • The temperature for the growth depends on the source of the cell as different organisms require different temperatures for cell growth and division. • Warm-blooded animal cells can be cultured at 37°C as the optimal temperature, whereas cold-blooded animals grow between 15°C-25°C.
• Primary cell culture • Primary cell cultures are obtained from fresh tissues that are removed from the organs with the help of an aseptic razor. • In some cases, the cells are removed by the use of chemical disintegrators or proteolytic enzymes. • The cell suspension obtained is washed with buffering liquid in order to remove the proteolytic enzymes. • The cell suspension is poured onto a flat surface which can be a culture vessel or a sterile Petri plate. • The cells that can adhere to the base of the vessel are overlaid with an appropriate culture medium and incubated at room temperature.
• Cell thawing • In the case of subsequent subcultures, the preserved cell culture might have to be used. • The water bath is heated to a temperature of 37°C, and the growth media where the cells are to be plated is warmed. • The warm medium is added to the culture vessel. The vial with the frozen cells is then placed in the water bath until thawed. • After thawing, the via is washed with 70% alcohol on the outside. The cell suspension is pipetted into the cell culture vessel and swirled gently to mix everything. • The medium is then incubated overnight under the usual growth conditions. The growth medium is replaced the next day.
• Trypsinizing Cells • Trypsinization is the method of separating adherent cells from the surface of the culture vessel with the help of proteolytic enzymes. It is done when the cells are to be used for passaging, counting, or other purposes. • The medium is removed, and the cells are recovered. The cells are then washed with phosphate buffer. • Warm trypsin-EDTA is added to the vessel so as to cover the monolayer. The vessel can be rocked to ensure that the monolayer is coated. • The vessel is incubated in a CO2 incubator at 37°C for 1-3 minutes. • The vessel is removed from the incubator, and the flask is firmly tapped on the side with the palm of the hand to assist detachment. • Once the cells are dislodged, they are resuspended in an appropriate growth medium containing some amount of serum. • The cells are then separated with the help of syringe needles by disrupting the cell clumps and used accordingly.
• Advantages • Animal cell culture makes the use of a low amount of reagents. • Serial passaging maintains the homogeneity of the cell types. • Provides controlled physiological conditions. • Provides controlled physiochemical conditions like temperature, oxygen concentration, ph etc. • Disadvantages • Animal cell culture requires high technical skills to interpret and to regulate the animal cell culturing. • It is a very expensive method to carry out.
• Applications of Animal cell culture • The following are some of the applications of animal cell culture; • a. Production of vaccines • b. Recombinant proteins • c. Gene Therapy • d. Model systems • e. Cancer Research • f. Production of Biopesticides •
Tissue culture •In vitro cultivation of organs, tissues & cells at defined temperature using an incubator & supplemented with a medium containing cell nutrients & growth factors is collectively known as tissue culture. •Different types of cell grown in culture includes connective tissue elements such as fibroblasts, skeletal tissue, cardiac, epithelial tissue (liver, breast, skin, kidney) and many different types of tumor cells.
Organoids • Organoids are three‐ dimensional (3D) miniaturized versions of organs or tissues that are derived from cells with stem potential and can self‐organize and differentiate into 3D cell masses, recapitulating the morphology and functions of their in vivo counterparts.
Properties • it has multiple organ-specific cell types; • it is capable of recapitulating some specific function of the organ (e.g. contraction, neural activity, endocrine secretion, filtration, excretion); • its cells are grouped together and spatially organized, similar to an organ.
Applications
Co2 incubator Dishes Centrifuges Autoclaves Vacuum Ovens ELISA readers
Intraperitoneal (IP) Injection* - *Angle*: 20–30° - *Site*: Lower right quadrant of the abdomen (to avoid vital organs). - *Volume*: Up to 5–10 mL/kg - *Notes*: Insert bevel up, tent the abdomen slightly if needed. *Subcutaneous (SC/SQ) Injection* - *Angle*: 30–45° - *Site*: Loose skin over the shoulders or flank - *Volume*: Up to 5 mL/site - *Notes*: Tent the skin and insert needle into the subcutaneous space. *Intravenous (IV) Injection* - *Angle*: 15–30° - *Site*: Lateral tail vein (commonly), can use other veins with training - *Volume*: Up to 5 mL/kg (slower rate) - *Notes*: Warm tail to dilate veins; ensure no leakage or swelling.
*Intramuscular (IM) Injection* - *Angle*: 45–90° - - *Site*: Quadriceps or gluteal muscles - *Volume*: Up to 0.1–0.3 mL/site - *Notes*: Use small-gauge needles; aspirate slightly to avoid vessels. . *Intradermal (ID) Injection* - *Angle*: 10–15° (very shallow) - *Site*: Usually back or flank - *Volume*: Very small, up to 0.1 mL - *Notes*: Should produce a small bleb/wheal.
END

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Final Term Syllabus.pptx model systems ppp

  • 1.
  • 2.
    Introduction • IBD isa group of chronic relapsing inflammatory conditions of the colon & small intestine • It is chronic gastrointestinal disorder characterized by intestinal inflammation and mucosal tissue damage initiated and perpetuated by a dysregulated immune response along with several intra- and extra intestinal manifestations, including autoimmune phenomena
  • 3.
    • 2 majormanifestations of IBD are  Ulcerative colitis  Crohn’s disease  https://www.youtube.com/watch?v=iefghc2g91M
  • 4.
    signs & symptoms • Diarrhea •Rectal bleeding • Passage of mucus • Abdominal pain • Anorexia • Fever • Weight loss Mechanisms of inflammatory bowel disease.
  • 5.
    • Mechanisms ofinflammatory bowel disease. (FOOTNOTE OF FIGURE) • The intestine shelters a large diversity of microbiota that are in perfect balance (symbiosis); • balance can be affected by many factors leading to the appearance of pathogenic bacteria that can alter the intestinal barrier and lead to the development of inflammatory bowel disease. • The stimulation of the mucosal immune system may occur as a result of the penetration of bacterial products through the mucosal barrier, leading to their direct interaction with immune cells, especially dendritic cells and lymphocyte populations, to promote a classic adaptive immune response. • Alternatively, bacterial products may stimulate the surface epithelium, possibly through receptors that are components of the innate immune-response system; the epithelium can, in turn, produce cytokines and chemokines that recruit and activate mucosal immune cells. • Activation of classic antigen-presenting cells, such as dendritic cells, or direct stimulation through pattern-recognition receptors promotes the differentiation of type 1 helper T-cells (Th1) in patients with Crohn's disease (shown here) or, possibly, atypical type 2 helper T-cells in patients with ulcerative colitis. The stereotypical products of Th1 promote a self- sustaining cycle of activation with macrophages. In addition to producing the key cytokines that stimulate Th1 (interleukin-12, interleukin-18, and macrophage migration inhibitor factor), macrophages produce a mix of inflammatory cytokines, including interleukin-1, interleukin-6, and most notably tumor necrosis factor, which target a broad variety of other types of cells. The latter include endothelial cells, which then facilitate the recruitment of leukocytes to the mucosa from the vascular space. Most important, these functions may be altered either by genetically determined variants, as exemplified by germ-line mutations in the gene encoding NOD2, the product of the IBD1 locus, in some patients with Crohn's disease, or by environmental factors.
  • 6.
    Screening Models Sodium trimethylsilylpropanesulfonate(DSS) Sodium trimethylsilyl propionate (TSP)
  • 7.
  • 8.
    Chemically induced colitis 1.Oxazolone induced • Oxzolone is haptenizing agent (To react an antigenic compound) • widely used to induce colitis in mice in order to evaluate the pathological processes involved in the perpetuation of ulcerative colitis. • This model of colitis generates an immune response mediated by Th2 cells • resembles human ulcerative colitis on the basis of manifestations of inflammation of the mucous membranes, epithelial microulcerations, and histopathological changes in the distal colon
  • 9.
    • Procedure :Enema (rectal administration) of oxazolone (3%) with ethanol would induce colitis it cause colitis earlier than other other chemicals Effects :Initial symptoms starts second day after the enema, and symptoms diminished after 10–12 days. Colitis, accompanied by ulcers, was localized in the distal colon.
  • 10.
    L-10 Knockout Modelfor IBD Interleukin-10 (IL-10) is a critical anti-inflammatory cytokine that regulates immune responses in the gut. Mice genetically engineered to lack IL-10 (IL-10⁻/⁻) spontaneously develop chronic enterocolitis resembling human IBD, particularly Crohn’s disease.
  • 11.
    Generation of theIL-10 Knockout Mouse •Strain: C57BL/6 or BALB/c background. •Genetic Engineering: Targeted deletion of the Il10 gene via homologous recombination in embryonic stem cells. •Breeding: Heterozygotes are bred to produce homozygous IL-10⁻/⁻ mice. Disease Manifestation •Spontaneous colitis appears typically after 3–8 weeks of age. •The severity may depend on: • Genetic background (e.g., C57BL/6 mice develop milder disease). • Microbiota composition (germ-free IL-10 KO mice do not develop colitis).
  • 12.
    Feature Description Histopathology Mucosal thickening, lymphocyticinfiltration, crypt abscesses, ulcerations Cytokine profile Increased TNF-α, IL-1β, IFN-γ Immune response Enhanced Th1 and Th17 responses Microbiota dependency Colitis is driven by commensal bacteria; germ- free mice remain disease- free Features of IBD in IL-10 KO Mice
  • 13.
    Applications in Research •Studyingpathogenesis of IBD. •Testing anti-inflammatory therapies (e.g., biologics, probiotics, dietary interventions). •Exploring the gut microbiome–immune system interaction. •Evaluating nutritional and environmental modifiers of IBD. Example Experimental Design
  • 14.
    Group Treatment Purpose WTcontrol No treatment Baseline comparison IL-10 KO No treatment Disease model IL-10 KO + Drug A Anti-inflammatory compound Therapeutic effect IL-10 KO + Probiotic Specific probiotic strain Microbiome-based intervention Study Plan
  • 15.
    Ethical Considerations •Mice developchronic inflammation and may require early endpoints or humane euthanasia. •Ensure IACUC approval or equivalent ethical clearance.
  • 16.
    Limitations of theIL-10 Knockout Model for IBD 1. Microbiota Dependency •Colitis does not develop in germ-free conditions; the disease requires colonization with commensal gut microbiota. •Disease severity and progression vary based on microbiota composition, making reproducibility across labs difficult. 2. Lack of Human IBD Complexity •IL-10 KO mice primarily exhibit Th1-mediated inflammation, which closely models Crohn’s disease, but not ulcerative colitis. •Human IBD is polygenic and influenced by multiple environmental and immune factors; IL-10 KO mice lack this multifactorial complexity. 3. Chronic, Non-Relapsing Disease •Unlike human IBD, which is characterized by relapsing-remitting cycles, the IL-10 KO model shows progressive, chronic inflammation without natural resolution phases.
  • 17.
    4. Systemic Effectsof IL-10 Deficiency •IL-10 has widespread anti-inflammatory roles beyond the gut; IL-10 KO mice may develop extraintestinal inflammation (e.g., arthritis, prostatitis), complicating interpretation of gut-specific effects. 5. Genetic Background Sensitivity •Disease severity is strain-dependent (e.g., more severe in BALB/c than C57BL/6), which can lead to inconsistent results unless carefully controlled. 6. Not Suitable for Late-Onset IBD •Inflammation typically appears at a young age (3–8 weeks), which doesn’t mimic adult-onset IBD seen in many human cases. 7. Non-targeted Immune Deficiency •Complete IL-10 knockout affects multiple immune cells (T cells, macrophages, dendritic cells), making it hard to isolate the effects of individual immune pathways
  • 18.
    Models of ChronicKidney Disease (CKD)
  • 19.
    Introduction to ChronicKidney Disease (CKD) • - Definition and staging (GFR < 60 mL/min/1.73 m² for >3 months) • - Pathophysiology: Nephron loss, fibrosis, inflammation • - Clinical relevance: Progression to End Stage Renal Disease
  • 20.
    Importance of CKDModels • - Study of renal mechanisms and pathophysiology • - Evaluation of new therapies and biomarkers • - Modeling comorbidities (e.g., diabetes, hypertension)
  • 21.
    In Vivo AnimalModels: Surgical • - 5/6 Nephrectomy: Simulates chronic renal failure • - Unilateral Ureteral Obstruction (UUO): Fibrosis model https://www.youtube.com/watch?v=CuT4kMW1c2c https://www.youtube.com/watch?v=z4MjyzlQc5Y
  • 24.
    In Vivo Models:Chemical & Genetic • - Adenine diet: Tubular injury and fibrosis • - Cisplatin/Aristolochic acid: Nephrotoxic injury • - Col4a3 KO (Alport), db/db (diabetic nephropathy)
  • 26.
    In Vivo Models:Hypertension-Induced • - Spontaneously Hypertensive Rats (SHR) • - DOCA-salt model: Hypertension and fibrosis
  • 27.
    In Vitro CKDModels • - Cell lines: HK-2, podocytes, mesangial cells • - Organoids: 3D nephron-like structures from stem cells • - Kidney-on-a-chip: Real-time, flow-based analysis Podocytes are highly specialized epithelial cells that cover the outside of the glomerular capillary. Mesangial cells are contractile cells which pack the glomerulus and have features of smooth muscle. HK-2 cells are derived from a normal, human adult male kidney. The cell line has applications in toxicology research.
  • 28.
    Model Selection Criteria •- Based on research goals: Fibrosis, inflammation, filtration defects • - Ethical/logistical considerations • - Relevance to human CKD and co-morbidities
  • 29.
    Limitations of CKDModels • - Species differences and lack of chronicity • - Often single-factor models • - Ethical concerns and cost
  • 30.
    Emerging and AdvancedCKD Models • - Humanized mice and iPSC-derived organoids • - CRISPR/Cas9 gene-edited models • - AI-based simulation and in silico predictions
  • 31.
    Conclusion • - Nosingle model is ideal—combine in vivo, in vitro, and in silico • - Model choice depends on the hypothesis and translational goals • - Future lies in personalized and human-relevant systems
  • 32.
    Animal Models ofDiabetes Mellitus
  • 33.
    Introduction • Diabetes Mellitusis a chronic metabolic disorder with two major types: • Type 1: Autoimmune β-cell destruction → Insulin deficiency • Type 2: Insulin resistance with progressive β-cell dysfunction • Animal models help in pathogenesis study, drug testing, and mechanism elucidation
  • 34.
    Ideal Model Characteristics •Reproduces key features of human diabetes • Predictive for therapeutic efficacy • Genetically manipulable and cost-effective • Reproducible with measurable biomarkers
  • 36.
    1. Surgical induced 5/7/2025 ●Partial pancreatectomy: usually anterior lobe is left intact Disadvantages ● Loss of α cells (glucagon and somatostatin loss) ● Loss of pancreatic enzymes (creates digestive problems) ● Difficult to achieve
  • 37.
    5/7/2025 • naturally occurringchemical • toxic to the insulin-producing beta cells of the pancreas • chemotherapeutic drug for treating certain cancers of the islets of Langerhans • used in medical research to produce an animal model for type 1 diabetes. • It is also an antibiotic effective against Gram-negative bacteria. Chemically induced Type 1 Diabetes Models- STZ
  • 38.
    5/7/2025 Mechanism ● Cell methylation ●Free radical generation ● Nitric oxide production
  • 39.
    5/7/2025 Procedure Male Wistar Rat- 150-200gm Group- Test, Standard, Control STZ. Dose- 60 mg/kg iv or ip ● Done in mice (175-200 mg/kg) and dogs (15 mg/kg for 3 days)
  • 40.
    14 5/7/2025 Evaluation ● Blood glucoselevel is estimated. ● Compare the result of test with standard and control.
  • 41.
    5/7/2025 ADVANTAGE • Selectivity towardsβ cells • Lower mortality rate • Longer diabetes induction Limitations • Rabbits and guniea pigs are resistant to its diabetogenic effect
  • 42.
    Type 1 DiabetesModels- NOD Mouse Non- obese diabeties • Spontaneous autoimmune β-cell destruction • Strong MHC II association (H2-g7) • Mimics human T1DM immunology • Limitation: no ketoacidosis
  • 43.
    Genetically induced Type2 Diabetes Models- ob/ob & db/db • ob/ob: Leptin deficiency → Obesity, insulin resistance • db/db: Leptin receptor defect → T2DM progression • Used for obesity- • Leptin is a hormone your body releases that helps it maintain your normal weight on a long-term basis. The level of leptin in your blood is directly related to how much body fat you have. Leptin resistance causes you to feel hungry and eat more even though your body has enough fat stores. linked T2DM studies
  • 44.
    17 5/7/2025 Principle ● Encephalomyocarditis virus(EMC-D) selectively infects and destroys pancreatic β-cell. ● Associated with chronic islet cell inflammation, elevation of islet cell antibody, and prolonged presence of viral RNA in the pancreas. ● Others: RNA picornavirus, Coxsackie virus Viral induced
  • 45.
  • 46.
    T2DM- Diet &Transgenic Models • High-fat diet induces metabolic syndrome and T2DM • HFD + STZ: Mimics human late-stage diabetes • Transgenic KOs: IRS1/2, GLUT4, PPARγ, RIP-DTR
  • 48.
    Limitations & Summary •No model captures all aspects of human diabetes • Rodent models lack chronic complications • Species-specific immune and metabolic differences • Use combined models for better translational relevance
  • 49.
    Animal Models in CardiovascularDisease (CVD) Research
  • 50.
    Introduction • Cardiovascular diseases(CVDs) are the leading cause of death globally. • Animal models are essential for understanding disease mechanisms and testing therapies. • CVD research includes studies on atherosclerosis, hypertension, heart failure, and arrhythmias.
  • 51.
    Criteria for anIdeal Animal Model • Mimics human pathology closely • Genetic and physiological relevance • Reproducibility and reliability • Ethical acceptability and cost-efficiency
  • 52.
    Small Animal Models– Mice • Transgenic and knockout models (e.g., ApoE-/-, LDLR-/-) • CRISPR/Cas9 for precise genetic editing • Advantages: genetic tools, cost-effective, short lifespan • Limitations: metabolic and size differences from humans
  • 53.
    Rats in CVDResearch • Models include Spontaneously Hypertensive Rat (SHR), Dahl salt- sensitive rat • Used for hypertension, heart failure, and vascular studies • Better physiological match for cardiovascular studies than mice
  • 54.
    Large Animal Models– Pigs • Close anatomical and physiological similarity to humans • Used in studies of coronary artery disease and ischemia-reperfusion • Ideal for testing surgical techniques and medical devices
  • 55.
    Canine Models • Historicallyused in arrhythmia and myocardial infarction research • Similar cardiac electrophysiology to humans • Use limited due to ethical concerns
  • 56.
    Non-Human Primates (NHPs) •Genetically and physiologically closest to humans • Used in advanced drug and gene therapy research • Expensive and subject to strict ethical regulations
  • 57.
    Induction Techniques inCVD Models • Surgical: coronary artery ligation, aortic banding • Dietary: high-fat diets in ApoE-/- mice • Chemical: isoproterenol, doxorubicin-induced models • Genetic: targeted mutations to study specific pathways
  • 58.
    Applications of CVDAnimal Models • Study of disease mechanisms like plaque formation • Drug development and safety assessment • Evaluation of new therapies (gene, cell, regenerative)
  • 59.
    Limitations and Challenges •Species-specific responses limit translational accuracy • Ethical concerns with certain models • High cost and technical demands of large animals
  • 60.
    Future Directions • Developmentof humanized mouse models • Use of iPSC-derived cardiac tissues for in vitro research • Integration of omics data and AI for predictive modeling
  • 61.
    Summary • Animal modelsare vital for CVD research. • Each model has unique strengths and limitations. • Future efforts focus on ethical, accurate, and translationally relevant models.
  • 62.
    Lecture 16 MICROBES OFMEDICAL SIGNIFICANCE BIOFILMS 6 2
  • 63.
    Bacterial Biofilms: Introduction •In recent time, microorganisms have been discovered to evolved various mechanisms of surviving a harsh environment; among which are production of biochemical compounds such as biofilm, biosurfactants, and enzymes. • Biofilms are self synthesized extrapolymeric slimy matrix that encloses a sessile microbial community whose cells are characterized by their attachment to either biotic or abiotic surfaces (Vasudevan, 2014). • Biofilm consist of either a single or multiple species of microorganisms (Flemming and Wingender, 2010). • Biofilms promote and regulate metabolic activities, Provision of nutrients and protection (Vasudevan, 2014). 6 3 https://study.com/academy/lesson/what-are-biofilms-definition- formation-examples.html
  • 64.
    6 4 Composition of Biofilm •Microbial cells • Extra polymeric substances, EPS (Oliveira and Cunha,2008) EPS provides essential nutrients, protection, architectural integrity, enables genetic and intracellular transfer (Donlan, 2000). Table 1: Composition of biofilm (Vasudevan, 2014). Do not memorize the matrix percentages S/NO Component Percentage of Matrix 1 Water Up to 97% 2 Microbial cells 2-5% 3 polysaccharides 1-2% 4 Proteins <1-2% (including enzymes) 5 DNA and RNA <1-2% 6 Ions Bound and free
  • 65.
    Biofilm formation  Biofilm formationbegins when microorganisms response to factors such as • Cellular recognition of attachment site, • Nutritional cues • Exposure of planktonic cells to sub-inhibitory concentration of antibiotics (Lieve van et al., 2012)  Stages in biofilm formation include, Adhesion/Attachment, adhesion of bacteria to a surface is a two step process; primary bacterial adhesion and secondary bacterial adhesion (Chelius and Duffy, 2013) Intracellular aggregation, formation of monolayer involving polysaccharide adhesin (PIA) similar to poly–N- acetylglucosamine (Helimann, 2011) Biofilm maturation, biofilm become complex, cells actively proliferating leading to increase in population density, Forms glycocalyx and mushroom- like structure (Chelius and Duffy, 2013; Niels et al., 2011). Biofilm dispersal, nutrients and Oxygen become depleted and biofilm begin to degrade and cells released from the mushroom like structure (Karatan and Watnick 2009; Hong et al. 2010; Rowe et al. 2010) 5
  • 66.
    Fig1 Stages inbiofilm formation. Adopted from Kirk E. Anderson Genetics and Microbial Ecology 66
  • 67.
    Quorum sensing andBiofilm formation 67 https://www.youtube.com/watch?v=mQ43fuJJW7M&t=1s
  • 68.
    Clinicial significance ofbiofilms 68 Bacterial Biofilms Antimicrobial tolerance Medical device related infections Chronic infections Modulation of immune response https://www.youtube.com/watch?v=aXb4Bg6DXE4 https://www.youtube.com/watch?v=0DSA_8t4-UA&t=48s
  • 69.
    Biofilm and antimicrobial resistance. •Agents may form complex with EPS (Romling and Balsallobre, 2012) • Poor penetration of biocide agent resulting from EPS (Manavathu and Vazquez, 2014). • Passage of genetic determinant via horizontal gene transfer (Juhas, 2013). • Expression of molecular efflux pump (Bueno, 2014) https://www.youtube.com/watch?v=8VZH4GZLWZc
  • 70.
    Medical device relatedinfections 70 • Most medical implants such as intravascular catheters, Devices use in orthopedic shunts, Stent, suture are susceptible to biofilm formation (Romling and Balsalobre, 2012). • Provide a reservoir for recurrent and at times life threatening bloodstream and urinary tract infections (Desai et al., 2014). • Biofilm causes device failure
  • 71.
    Biofilms and chronicdiseases  Chronicity in diseases such as diabetic chronic wounds, • Cystic fibrosis, • Bronchopneumonia, • Otitis media, • Osteomyelitis (Bjarnsholt et al., 2009; Homoe et al., 2009; Tacconelli et al., 2009)  Biofilm triggers proinflammatory responses harmful to host tissues.  Responsible for delayed healing in diabetic wound (Zhao et al., 2013). 12 https://www.youtube.com/watch?v=dws3bEj2Cjg
  • 72.
    Modulation of immune response EPScomponent(s) at times triggers proinflammatory response that fails to recognize host tissue as ‘self’ with detrimental effects (Nibali et al., 2014). Microbial biofilm mediated inflammatory response halts the chronic wound healing process at the inflammatory stage and prevent proliferation and healing (Manavathu and Vazquez, 2014). Lipopolysaccharides released from dental plaque initiate imflammatory immune response which triggers the release of inflammatory mediators (Benakanakere and Kinane, 2012). Bacteria-in-wound protected by biofilm from the host immune system. 13
  • 73.
    Future direction forcombating biofilm menace • Development of antibiofilm antimicrobial agents directed to targeting one or more of the major stages of biofilm development. • Extra polymeric matrix busting’ antibiofilm drug should have the ability to disrupt existing biofilm. • The use of bacterial/fungal adhesion resistant material for the construction of medical devices associated with biofilm. • Development of vaccines directed to targeting quorum sensing. 14
  • 74.
    7 4 Lecture 17 Synthetic BiologyFor Clinical Research
  • 75.
     Synthetic Biology • Designing/redesigningnew biological parts, devices and systems that do not exist in the natural world. 7 5 • The term appeared in the literature in 1980 to describe bacteria that had been genetically engineered using recombinant DNA technology. • Synthetic biology = Bioengineering. • The final goal is to be able to design biological systems in the same way engineers design electronic or mechanical systems.
  • 76.
      Potential applicationsof synthetic biology range very widely across scientific and engineering disciplines, from medicine to energy generation. For example, designed microorganisms might be capable of producing pharmaceutical compounds that are extremely challenging for existing methods of chemical or biological synthesis. What can synthetic biology achieve? 76 https://www.youtube.com/watch?v=rD5uNAMbDaQ
  • 77.
     • Bacteria orviruses could be programmed to identify malignant cancer cells and deliver therapeutic agents (Serrano 2007). • Viruses have also been engineered to interact with HIV-infected cells, which could prevent the development of AIDS (De Vriend 2006). Medical Applications • Construction of an artificial metabolic pathway in E. coli and yeast to produce a precursor (arteminisin) for an antimalarial drug (Martin et al. 2003, Ro et al. 2006).
  • 78.
    • Biofuel: Thereare many ways of engineering microorganisms to produce carbon-neutral (or more environmentally friendly) sources of energy. For example, bacteria could be engineered to synthesize hydrogen or ethanol by degrading cellulose, although further work is needed to overcome technical barriers. 78 • Synthetic vaccines. The fact that synthetic biology can ‗start from scratch‘ means that new synthetic vaccines could be produced in response to viruses that themselves evolve rapidly, such as those that cause severe acute respiratory syndrome (SARS) and hepatitis C (Garfinkel et al. 2007).
  • 79.
      There aretwo factors which make the risk governance of synthetic biology potentially problematic. • The first is that synthetic biology (like genetic engineering) involves the production of living organisms, which by definition are self- propagating. • The second is that with the growth of the Internet and the routinisation of many biotechnological procedures, the tools for doing synthetic biology are readily accessible (Garfinkel et al. 2007). Risks related to synthetic biology 79
  • 80.
    Ethical Issues –A certain Concern  Statements to the effect that the next 50 years of DNA evolution will take place ―not in Nature but in the laboratory and clinic‖ (Benner 2004:785), accompanied by inventions such as plants that produce spider silk, clearly challenge everyday understandings of nature and our place in it. 39 • Additionally, the flexibility of synthetic biology means that microorganisms could be created which are radically different from existing ones, and these microorganisms might have unpredictable and emergent properties (Tucker and Zilinskas 2006), making the risks of accidental release very difficult to assess in advance (De Vriend 2006).
  • 81.
    Cell culture asModels system
  • 82.
    Cell culture  Cellculture is the process by which prokaryotic, eukaryotic or plant cells are grown under controlled conditions. But in practice it refers to the culturing of cells derived from animal cells.  Cell culture was first successfully undertaken by Ross Harrison in 1907  Roux in 1885 for the first time maintained embryonic chick cells in a cell culture.
  • 83.
    Why is cellculture used for? Areas where cell culture technology is currently playing a major role. Model systems for Studying basic cell biology, interactions between disease causing agents and cells, effects of drugs on cells, process and triggering of aging & nutritional studies Toxicity testing Study the effects of new drugs Cancer research Study the function of various chemicals, virus & radiation to convert normal cultured cells to cancerous cells
  • 84.
    Types of cellculture • Animal cell cultures can be divided into two distinct groups depending on the number of cell divisions occurring during the process; 1. Primary cell culture •Primary cell culture is the first culture obtained directly from animal tissue via mechanical and chemical disintegration or enzymatic methods. •The cells of the primary cell culture are slow-growing cells that carry all the characteristics of the original tissue or cells. •Primary cell cultures can be subcultured to obtained other cultures that either continue to grow indefinitely or die after a few subcultures.
  • 85.
    Types of Animalcell culture • 1. Primary cell culture
  • 86.
    • Primary cellcultures can be further divided into two groups depending on the kind of cells present in the culture; • a. Anchorage-dependent/Adherent cells • The cells in the culture require a stable biologically inert surface for adherence and growth. • The surface should be solid and nontoxic as these cells are difficult to grow as cell suspensions. • These cells are usually obtained from the tissues of organs where the cells remain immobilized within the connective tissue. • Examples of adherent cells include kidney cells and mouse fibroblast STO cells.
  • 87.
    • b. Anchorage-independent/Suspension cells • These cells can grow efficiently as cell suspensions and do not require a solid surface for attachment. • These can be grown on liquid media continuously to obtain fresh subcultures. • The ability of the cells to grow as suspension depends on the source of cells as cells that remain as suspensions in the body are effective suspension cells. • Examples of suspension cells include blood cells that are vascular and remain suspended in the plasma.
  • 88.
    • 2. Secondarycell culture • Secondary cell cultures are obtained after the primary cell cultures are subsequently subcultured over a period of time in fresh culture media. • The cells of the secondary cell cultures are long-lasting as these have a higher lifespan due to the availability of appropriate nutrients at regular intervals of time. • Secondary cell cultures are favored over primary cell cultures as these are more readily available and are easy to grow and preserve. • The cells of the secondary cell culture might not resemble that on the parental tissue as mutations, and genetic alterations might be introduced during the subculture process.
  • 90.
    • 3. CellLines • A cell line is a group of cells that are formed from the subculture of primary culture consisting of a pure culture of cells. Cell lines usually display functional features that are close to the primary cells, but the genotype and phenotype of the cells can be modified. A cell line consists of several cell lineages with similar or different phenotypes. • Cell lines can be further divided into two groups based on the growth patterns of the cells;
  • 91.
    a. Finite celllines • Finite cell lines are cell lines where the cells in the culture divide for a limited number of times, after which they eventually die. • The cells in the finite cell lines can divide from 20 to 100 times before they eventually die and cannot divide anymore. • The number of cell division and lifespan depends on a number of factors like cell lineage differences, species, culture conditions, and media. • The cells of the finite cell lines grow as adherent cells on solid surfaces.
  • 92.
    • b. Continuouscell lines • Continuous cell lines are cells that exhibit indefinite growth via subsequent subcultures. • The cells in the continuous cell lines grow faster to form an independent culture. The cells are immortal and can divide indefinitely. • The cells in the continuous cell lines can be transformed via genetic alterations and are also tumorigenic. • The transformed cells are formed from the normal primary cell cultures after treatment with chemical carcinogens or by infection with oncogenic viruses. • The cells are capable of growing to prepare higher cell density and can grow as suspensions on liquid media. • These cells can even grow on top of each other to form multilayered structures on the culture vessels.
  • 94.
    • Examples ofcommon Cell Lines • The following are some of the common examples of cell lines; • a. HeLa cell line • HeLa cells are one of the first continuous culture human cell lines with the help of cells of the cervical carcinoma. • These cells are used for processes like virus cultivation and preclinical drug evaluation. • b. HL 60 (Leukemia) • c. MCF-7 (breast cancer cells)
  • 95.
    Confluency • Confluence （Confluent monolayer ） is when the adherent cells cover the adherent surface of the culture vessel. When culturing adherent cells, the rate of cell growth can be determined based on the ratio of the cultured cells covering the adhesion surface of the culture vessel (cell occupied area ratio or confluency).
  • 96.
    • Procedure orProtocol of Animal cell culture • 1. Growth Conditions • Animal cell culture requires the use of specific culture media that are more complex and specific than the basic culture media used for microbial growth. • Some of the important basic components of the media are inorganic salts, nitrogen source, energy source, vitamins, fat and fat-soluble vitamins, growth factors, and hormones. In some cases, pH buffering systems and antibiotics are also added. • The temperature for the growth depends on the source of the cell as different organisms require different temperatures for cell growth and division. • Warm-blooded animal cells can be cultured at 37°C as the optimal temperature, whereas cold-blooded animals grow between 15°C-25°C.
  • 97.
    • Primary cellculture • Primary cell cultures are obtained from fresh tissues that are removed from the organs with the help of an aseptic razor. • In some cases, the cells are removed by the use of chemical disintegrators or proteolytic enzymes. • The cell suspension obtained is washed with buffering liquid in order to remove the proteolytic enzymes. • The cell suspension is poured onto a flat surface which can be a culture vessel or a sterile Petri plate. • The cells that can adhere to the base of the vessel are overlaid with an appropriate culture medium and incubated at room temperature.
  • 98.
    • Cell thawing •In the case of subsequent subcultures, the preserved cell culture might have to be used. • The water bath is heated to a temperature of 37°C, and the growth media where the cells are to be plated is warmed. • The warm medium is added to the culture vessel. The vial with the frozen cells is then placed in the water bath until thawed. • After thawing, the via is washed with 70% alcohol on the outside. The cell suspension is pipetted into the cell culture vessel and swirled gently to mix everything. • The medium is then incubated overnight under the usual growth conditions. The growth medium is replaced the next day.
  • 99.
    • Trypsinizing Cells •Trypsinization is the method of separating adherent cells from the surface of the culture vessel with the help of proteolytic enzymes. It is done when the cells are to be used for passaging, counting, or other purposes. • The medium is removed, and the cells are recovered. The cells are then washed with phosphate buffer. • Warm trypsin-EDTA is added to the vessel so as to cover the monolayer. The vessel can be rocked to ensure that the monolayer is coated. • The vessel is incubated in a CO2 incubator at 37°C for 1-3 minutes. • The vessel is removed from the incubator, and the flask is firmly tapped on the side with the palm of the hand to assist detachment. • Once the cells are dislodged, they are resuspended in an appropriate growth medium containing some amount of serum. • The cells are then separated with the help of syringe needles by disrupting the cell clumps and used accordingly.
  • 100.
    • Advantages • Animalcell culture makes the use of a low amount of reagents. • Serial passaging maintains the homogeneity of the cell types. • Provides controlled physiological conditions. • Provides controlled physiochemical conditions like temperature, oxygen concentration, ph etc. • Disadvantages • Animal cell culture requires high technical skills to interpret and to regulate the animal cell culturing. • It is a very expensive method to carry out.
  • 101.
    • Applications ofAnimal cell culture • The following are some of the applications of animal cell culture; • a. Production of vaccines • b. Recombinant proteins • c. Gene Therapy • d. Model systems • e. Cancer Research • f. Production of Biopesticides •
  • 102.
    Tissue culture •In vitrocultivation of organs, tissues & cells at defined temperature using an incubator & supplemented with a medium containing cell nutrients & growth factors is collectively known as tissue culture. •Different types of cell grown in culture includes connective tissue elements such as fibroblasts, skeletal tissue, cardiac, epithelial tissue (liver, breast, skin, kidney) and many different types of tumor cells.
  • 103.
    Organoids • Organoids arethree‐ dimensional (3D) miniaturized versions of organs or tissues that are derived from cells with stem potential and can self‐organize and differentiate into 3D cell masses, recapitulating the morphology and functions of their in vivo counterparts.
  • 104.
    Properties • it hasmultiple organ-specific cell types; • it is capable of recapitulating some specific function of the organ (e.g. contraction, neural activity, endocrine secretion, filtration, excretion); • its cells are grouped together and spatially organized, similar to an organ.
  • 105.
  • 106.
  • 107.
    Intraperitoneal (IP) Injection* -*Angle*: 20–30° - *Site*: Lower right quadrant of the abdomen (to avoid vital organs). - *Volume*: Up to 5–10 mL/kg - *Notes*: Insert bevel up, tent the abdomen slightly if needed. *Subcutaneous (SC/SQ) Injection* - *Angle*: 30–45° - *Site*: Loose skin over the shoulders or flank - *Volume*: Up to 5 mL/site - *Notes*: Tent the skin and insert needle into the subcutaneous space. *Intravenous (IV) Injection* - *Angle*: 15–30° - *Site*: Lateral tail vein (commonly), can use other veins with training - *Volume*: Up to 5 mL/kg (slower rate) - *Notes*: Warm tail to dilate veins; ensure no leakage or swelling.
  • 108.
    *Intramuscular (IM) Injection* -*Angle*: 45–90° - - *Site*: Quadriceps or gluteal muscles - *Volume*: Up to 0.1–0.3 mL/site - *Notes*: Use small-gauge needles; aspirate slightly to avoid vessels. . *Intradermal (ID) Injection* - *Angle*: 10–15° (very shallow) - *Site*: Usually back or flank - *Volume*: Very small, up to 0.1 mL - *Notes*: Should produce a small bleb/wheal.
  • 109.