Allogeneic MCSs to make Cartilage for Knee Function

Allogeneic MCSs to make gristle for human knee joint Function accession 1.1 What is degenerative arthritis?Articular gristle is a highly resilient hyaloid meander composed of chondrocytes and surrounded by extra mobile phonephoneular telephoneular matrix express in a go which act as shock absorber, protects the swot from the friction and wear and helps in smooth move handst of the joint (Bhumiratana et al. 2014). osteoarthritis is a disease of joint where lack of gristle causes musculoskeletal distract and restriction of the movement or disability of the joint for the affected role. (Ahmed and Hincke, 2010) (Duthey, 2015).Reasons for cartilage deadening atomic get along 18 The impact / blow caused during sport activities or accident endure and tear because of overuse of a joint (Observed in elderly population) lack of movement (Medical News Today, 2017) foretell No.1. degenerative joint disease Affected Region go for witnesser www.osteoosteoOsteoarthritisresearc huk.orgOsteoarthritis foot affect every joint express in the form. As the knee-joint Osteoarthritis is the more or little common type of Osteoarthritis, in this report, we impart discuss about knee-joint Osteoarthritis only.Tibiofemoral and patellofemoral ar the two articulary pops that the knee consists of. As it shadower be seen in the below image, the modify cartilage, reduces the gap between joint and friction is generated between the overdresss which in conclusion results in bone erosion and causes muscle upset or discharge or restriction to the movement.Figure No.2. Osteoarthritis affected Knee depict Source http//www.bupa.co.uk/health-information/directory/o/OsteoarthritisOsteoarthritis is estimated to affect 250 one thousand million people worldwide. Osteoarthritis sufferers include men and women, children and adults. And according to World Health Organization, 30% of men and women over the age of 65 nonplus Osteoarthritis (Woolf and Pfleger, 2003). Worldw ide, 9.6% of men and 18.0% of women over the age of 60 years take a crap symptomatic Osteoarthritis. close to 80% of those with Osteoarthritis depart have limitations in movement, and 25% hind endnot carry out their major activities of daily life (Duthey, 2015).Figure No.3. Prevalence of Osteoarthritis of KneeImage Source Burden of major musculoskeletal conditions, Bulletin of the WHO 20031.2 Treatments accessible for Osteoarthritis on that point ar confused ways to cure Osteoarthritis when it is at the initial level, such(prenominal) as Exercise and weight dischargeBracingMedicationViscosupplementationNutritional supplements (Duthey, 2015). moreover when it becomes incurable by exploit and medication, surgical operations must be performed. Surgical procedures include Debridement i.e. Smoothening of the cartilage using surgical instrumentsMarrow Stimulation, a extendment which helps in re harvest of cartilage in the joint (but this process is less reliable) (Treatmen t Options for Osteoarthritis in the Knee, 2017).Mosaicplasty, a process where the cartilage from some separate joint of system is used. But this process has size limitations (Medical News Today, 2017).Autologous Chondrocyte Implantation, a interposition in which a sm alone part of no-load bearing cartilage is removed from the joint of the patient by Arthroscopy, regrown and multiplied in the laboratory and then implant back in the body by a procedure c in ei in that locationd arthrotomy. (Cartilage Repair, 2017) (Ahmed and Hincke, 2010) (Duthey, 2015). point though the Autologous Chondrocyte Implantation seems effective and easy, it has many disadvantages such as The patients cartilage sample must be removed by a medical procedure, marked/tagged and treated separately just standardised blood sample.This treatment requires big Logistics and impart Chain.It requires a lap of quantify (approximately 6 weeks) for carrels to multiply. Hence, till then the patient volition suf fer from pain (Peretti et al. 2000).1.3 Proposed Treatment for Osteoarthritis All these problems commode be solved by Allogeneic gentlemans gentleman Mesenchymal straw prison jail electric cell. For autologous transplant bestower and receiver atomic number 18 same, whereas for allogeneic transplant, the donor and the receiver atomic number 18 different. The selection of the donor must be done c atomic number 18fully cause if the thread type, i.e. HLA (Human Leukocyte Antigens) doesnt match, the patients body allow for treat the transplanted organ or create from raw material as a foreign body. It cogency result in GVHD i.e. Graft Vs Host infirmity. It is a flumpal tolerant system response against stem cell transplant (Si et al. 2011).Selection of donor for allogeneic transplant Syngeneic (i.e. Twins) It is the perfect HLA match, but very few people have a twin.HLA- matched relative (sibling) It is the second preferred option as HLA leave be closely matched.HLA-matched unrelated donor, it female genitalia be possible to discern a donor whose HLA matches to the patient.HLA-mismatched family member, even though the HLA doesnt match, it has great chance that patients body may accept it.Umbilical cord blood, stem cells retrieved during birth of the patient and preserved in a cell intrust. It leave behind be safest of all but stem cells must be available (Flomenberg et al. 2004).Hence, allogeneic implant will make sure that the patient fashion have to undergo two medical procedures, as seen in autologous chondrocyte implantation.1.4 What ar HMSCs?HMSC means Human Mesenchymal Stem kiosks. They argon multipotent cells, which have the ability to transform into bone, muscle, fat or cartilage, etc. upon the proper simulation of providing environmental conditions in the laboratory. They have potential for regeneration (Si et al. 2011) (Li, LHeureux and Elisseeff, 2011) (Wei, 2013).Figure No.4. Potential of MSCsImage Source http//www.medicalnewstoda y.com/articles/241215.phpFigure No.5. Mesenchymal Stem stallsImage Source http//www.cytopeutics.com/IntroductionOfStemCells.htmlFor knee restoration, cartilage cells are needful. Hence, the MSCs will be simulated for cartilage development. MSCs exists in almost all tissues. These cells can be easily obtained from bone marrow, adipose tissue, cord cells and molar cells, foetal liver, muscle, and lung (Ahmed and Hincke, 2014) (Si et al. 2011).1.5 Product delivery to the forbearing For blood trans confederacy, the blood group and front of Rh factor is checked and the matching blood is introduced into the body. Similarly, after checking the tissue (HLA) match, the best matching cells are chosen and regrown exponentially in the controlled environment of a laboratory. When the required number of cells, shape, and size is achieved, the cartilage is implanted into the patient via an open joint surgery named arthrotomy. This implanted cartilage will puzzle out exactly as that of the o riginal cartilage. This cartilage will black market competencyily for approximately 10 years (Ahmed and Hincke, 2010).1.7 Functioning of the product in the patients body Since, the HLA was matched, and the cartilage is manufactured using MSCs which has the same functional properties and characteristics that of the original cartilage, the function of the joint will return to normal. There wont be any complication after the treatment and that graft will be certain by the body as a part of it, it wont be treated as a foreign body.MANUFACTURING FEASIBILITY REVIEW 2.1 catamenia Manufacturing Technology and Scope for Future Currently, the knee restoration is done via other surgical procedures. But because of those procedures have many limitations and they give only fly-by-night relief, allogeneic Mesenchymal Stem Cell therapy will replace them in the coming time. Mesenchymal Stem Cell therapy is currently under development. Various tests are being performed on them in the laborator y (Ahmed and Hincke, 2010).First, the bone marrow or adipose tissue or cord sample is collected from the donor. wherefore the mesenchymal stem cells are separated out from other cells, such as fat or muscle by centrifugation or apheresis. These two density separation processes are feasible only for liquid. For the extraction from solid tissues, the slices of tissue are digested by the enzymes such as trypsin or collagenase. It breaks the bonding of cells i.e. the extracellular matrix (ECM) that holds the cells. Hence, the cell line is piece (Li, LHeureux and Elisseeff, 2011).Then the cells are harvested. During the cell polish process, at that place are various parameters that need to be monitored, little inconsistency will result in subnormal product or it might be just a waste of product. Temperature, humidity, oxygen, pH level of the cell culture reagent, nutrient supply and waste removal are the physical parameters and cell count and cell viability are the biological paramet ers that need to be monitored (Schwamb, Puskeiler and Wiedemann, 2015). erst the desired number of cells is achieved, boundary for CMB (Condensed Mesenchymal Cell Bodies) is set. Then the cells are condensed to increase the seeding density as the cartilage requires higher seeding density. Then the fusion of the CMB happens. Now this fused CMB is pressurized against a porous decellularized bone matrix to create dense cellular region i.e. cartilage (Bhumiratana et al. 2014). As the knee joint is a mechanical tissue, physical stimulation is needed for its development. However, excessive stimulation can lead to cartilage damage (Ahmed and Hincke, 2010). Cartilage then sticks to the surface of that bone matrix and takes its shape while growing around it. Then it is removed from the bone matrix to implant into the knee joint of the patient (Bhumiratana et al. 2014).Figure No.6. Condensed Mesenchymal Cell Bodies FusionImage Source http//www.pnas.org/content/111/19/6940.abstractCurrently, only culture plates and culture flasks are being used for allogeneic Mesenchymal Stem Cells as it is still in interrogation configuration (Schwamb, Puskeiler, and Wiedemann, 2015).Figure No.7. Culture flasks and platesImage Source https//www.shutterstock.comBut monitoring all these parameters becomes very hard when using flasks and plates. And the cells need to be shifted into bigger containers frequently. Also, flasks and plates are not useful for mass production because of size limitation and economical consideration. Hence, a device named bioreactor can replace them and still perform all those tasks efficiently.Figure No.8. Bioreactor for mass Cell CultureImage Source http//www.bioc.rice.edu/bios576/nih_bioreactor/NDL_Bioreactor%20Page.htmlIt is a container which is feasible for both aerobic and anaerobic cell culture and can be used for suspended as well as immobilized cells (Sandhya Anand, 2017). It can be operated in batch, fed batch and continuous mode. As MSCs are sur face anchorage dependent, the extra agitation or stirring might result in damage to the tissue. And the MSCs require oxygen to grow, so it will be an aerobic, immobilized, batch production bioreactor. (Martin, Wendt and Heberer, 2004) (Oragui, Nannaparaju and Khan, 2011).2.2 Challenges in mass production of MSCs striking scale in vitro expansion of MSCs is very complex because reserveing cells quality attributes such as identity, potency, purity and safe is extremely hard. It is hard to monitor that the cells are not undergoing any quality changes while expansion and harvesting.Another argufy is obtaining required no of cells and their recovery.MSCs are not suspension type, but anchorage dependent therefore the surface area for anchorage and proliferation must be taken into account.As allogeneic treatments are supposed to be for a lot of people, hence the required no of cells must be extremely large.There are 3 major and 3 minor types of HLAs in MHC class I and 3 major and 2 m inor types of HLAs in MHC fellowship II. So, there are lots of variants to manufacture and maintain for the cartilage manufacturer.2.3 clinical Demand for Dosage Even though there are 250 million people suffering from Osteoarthritis and 3.6% of them are suffering from knee Osteoarthritis i.e. 9 million people. More than 600,000 knee replacements are performed each year in the United States alone (A Nation in Motion, 2017). In UK 160,000 knee replacement surgeries are performed every year (Joint replacement statistics, 2017).As the cartilage manufactured in the laboratory exhibit almost similar properties that to the inherent cartilage, it is expected to last approximately 50-60 years (i.e. Average human life) if there are no unexpected tragedies. Hence, once treated properly, the patient wont have to worry about the joint in his life again.2.4 Supply Chain for Product Figure No.9. Formation of win Cell beachFirst the cell line is chosen for culturing, it can be a well-known cel l line or a newly found cell line. After certain passages, when the desired number of cells is achieved, the Master Cell Bank will be established. In this case, many Master Cell Banks are needed as there are many types of tissues. Then one portion of master cell bank will be used for research purpose, i.e. the working cell bank and the rest will be cryopreserved for future use. Good manufacturing practice protocol should be followed during cell culturing.Figure No.10. Clinical parade for Cell CulturingThe working cell bank will be used for manufacturing of cells for mass production after interrogatory is performed. Several production runs (i.e. races) will be performed to obtain the required number of cells. Then the cells will be cryopreserved in central storage and distributed via local anesthetic channels until there is a patient who needs them.2.5 Risk assessment The main aim of risk assessment is to prevent transmission of diseases, and evacuate harm to individuals and the environment. In many countries, the performance of risk assessment is a legal requirement. (University of Manitoba)RiskImpactProbability of OccurrenceMitigation dodgingTissue/cell originRejection of Cells startThorough scrutiny of cell lineLack of Donor HistoryTransmission of DiseaseLowChoosing a donor carefullyMismatch of HLAGraft vs Host DiseaseIntermediateCareful matching of HLAEnvironmental ChangesChange in cell QualityHighClose monitoring of environmental conditionsPlasma Derived veridicalCell line contamination with unwanted cellsHighProper filtration of MSCs(Herberts, Kwa and Hermsen, 2011)2.6 Biosafety verse Depending upon the as the product is human derived, Biosafety Level 2 practices, equipment and facilities are chosen. It is most suitable for clinical, diagnostic and teaching purposes.Laboratory personnel must maintain hygiene while entering and exiting the lab. Decontaminated of potentially infectious material must be done before disposal, either by a disinfectant , or by autoclaving. Personal protective equipment is only required when there is a possibility of exposure to hazardous material. The laboratory must be stranded from the general building. Laboratory personnel must be trained in handling pathogenic agents. Access to the laboratory should be limited during the work. accredited procedures in which infectious aerosols or splashes may be created biological safety cabinets or other physical containment equipment should be used and the rest can be performed on the open bench. Biosafety level 2 is suitable for autochthonal moderate-risk agents. This includes various microbes that cause mild disease to humans, or are nasty to contract via aerosol in a lab setting, human derived blood, body fluid, tissues, or primary cell lines (Inc, 2017).PROCESS MAP AND CELL yield ANALYSIS 3.1 Process Map Figure No.11. Process Map for HMSC TherapyProcess Description Cell lines are created/chosen for each type of tissue (HLA).Shipping of the tissue sam ple to cell therapy processing facility.HMSC isolation and culturing in culture chambers (manual production using culture flask or culture plate) or bioreactor is performed.Fresh HMSCs are then tested for various parameters such as identity, potency, purity and safety, the modifications are done.Aliquoting of HMSC samples (i.e. Master Cell Bank) is done. halt and storage at -196 C in Vapor Liquid northward (i.e. Cryopreservation) for future reference and use is done (Inc, 2017).Cells are thawed i.e. their temperature is brought up to normal room temperature and further increased to 37 C (Normal frame Temperature) for best cell growth result (Inc, 2017).Cell characterization per reveal Criteria for mellow outed HMSCsExpansion of thawed HMSCs using an incubator and/or bioreactor for production.energizing of HMSCs into final cell therapy product.Shipping of final product to medical treatment centre.Implantation of the cartilage into the patient by open joint surgery, i.e. arthrotomy (Harel, 2013).Cell harvest-home Analysis As there are many types of tissues (HLA), testing for all of them must be performed and validated. Hence, the whole process will be repeated several times for each type of cells. comment info Desired seeding density= 1 million/mlDuration of departure= 72 hoursDoubling epoch= 36 hoursEfficiency= 80% (Average efficiency) commentary Vial contains= 1.00E+09 cellsDose per Patient= 1.00E+09 cells =1 vial of dose harvest-home Rate= Ln (2) /Doubling Time= 0.019254Seeding Density1,000,000 pass Duration72Doubling Time36Efficiency0.8Input Vial1.00E+09Growth Rate0.019254 course 115Patients flaskDose Per Patient1.00E+09T25MCB CreationReal SAInputIdeal SA return respectT75Thaw1.00E+09800.008.00E+08All Flask of Same SizeT175Passage 17006.40E+082560.002.56E+094*T175T500Passage 225002.05E+098192.008.19E+095*T500T650Passage 378006.55E+0926214.402.62E+106*T1300T1300Passage 4260002.10E+1083886.088.39E+101*T26000T3250T6500T26000MCBs Created21.51Equivalent Vi als83.89Cells Per 5-Layer flask3.90E+09Phase 1Real SAInputIdeal SAOutputNoteThaw3.99E+093195.663.20E+09Passage 130002.56E+0910226.111.02E+106*T500Passage 291008.18E+0932723.563.27E+107*T1300Dosages3.27E+012.62E+01For Phase 1 Testing21 Master Cell Banks will be created in a 5-layer flask (T3250). It would be similar to the size of 83.89 input vials after 4 passages. From those 21 cell banks, 1 will be thawed and the rest will be cryopreserved. That 1 cell bank will be chosen as working cell bank and will be harvested for production.During Phase 1, treating 30 patients will be the target. Hence, 30 vials of doses should be manufactured during degree one. both time 20% loss of cells is considered while changing the flask.And During passages, exponential growth will take place. Formula for Exponential Growth is The Ideal surface area is calculated by Flask size was kept equal during every passage. And Actual Surface Area was always chosen less than Ideal Surface Area to maintain the desired density and environment.Flask of capacity 5-Layer was chosen for MCB creation.Calculations for MCB,Number of doses,After successful testing of phase 1, phase 2 will bring where 300 patients will be treated. So, 300 vials of cells will be required.PHASE 2Real SAInputIdeal SAOutputNoteThaw3.99E+093195.663.20E+09Passage 130002.56E+0910226.111.02E+106*T500Passage 291008.18E+0932723.563.27E+107*T1300Passage 3325002.62E+10104715.391.05E+115*T6500Passage 41040008.38E+10335089.263.35E+114*T26000Dosages3.35E+02For Phase 2 TestingAfter successful testing of phase 1 and phase 2, phase 3 will begin when mass production will start and 100s of 1000s of people will be treated with allogeneic HMSC derived cartilage.PHASE 3Real SAInputIdeal SAOutputNoteThaw3.99E+093195.663.20E+09Passage 130002.56E+0910226.111.02E+106*T500Passage 291008.18E+0932723.563.27E+107*T1300Passage 3325002.62E+10104715.391.05E+115*T6500Passage 41040008.38E+10335089.263.35E+114*T26000Passage 53120002.68E+111072285.631 .07E+1212*T26000Passage 610660008.58E+113431314.003.43E+1241*T26000Passage 734060002.75E+1210980204.811.10E+13131*T26000Dosages1.10E+04For Phase 3Since, there are 7 passages the process to manufacture 11000 vials will require approximately 25 (considered an extra time for changing flask) days. And at that rate 15 batches will be produced per year and approximately 165000 patients can be treated per year.As there are 6 types of tissues (HLA) wide number of patients treated will be 990000 approximately. It will be equivalent to 11% of global demand.Using Bioreactor for Phase 3 Instead of using Culture flasks or plates, a bioreactor can be used for cell culturing. To check which of these two techniques will be more efficient, all the parameters are kept same. And total time of 7 passages will be considered as one passage time for bioreactor.Passage Duration504Doubling Time36PHASE