Cell and Tissue Engineering Flashcards
Give a short timeline of the development of regenerative medicine
1960’s - first adult stem cell (mouse) isolated
1981- Mouse embryonic stem cells isolated
1998 - Human ESCs isolated
2007- Human iPSCs cultivated
Give reasons for why regenerative medicine is required
People living longer
- Global median age 26 in 1998, 38 by 2050
- European median age 47.4 by 2050 (1 in 3 people over 60)
Therefore many age related disorders and degenerative diseases
Give examples of current therapies
Inert Biomaterials
- Metal joint replacement, dentures, heart valves
- Complications: failure, loosening, no growth or remodelling, revision surgery often needed
Cell based therapies
Autologous - patients own cells and tissues. (skin grafts etc.) Complications of pain, harvest site morbidity, infection and cost
Allogeneic - Cells and tissues from another donor. Complications: infection, immune rejection, donor shortage
Define Tissue egineering
An inter-disciplinary field that applies the principles of engineering and life sciences toward the development of substitutes that reserve , maintain or improve tissue function or a whole organ
Define Cell Engineering
The process of modifying cells for therapeutic application. May involve genetic, mechanical, chemical modification and reprogramming
State the source and methods of obtaining and culturing primary cells
Established from a tissue source (biopsy etc.)
Can be isolated in explant culture (samples grown on plastic surfaces) or before culture by breaking up the tissue
Cells can be separated by cell surface marker protein expression or density centrifugation
Growth conditions are anchorage dependant using growth medium
State the source and methods of obtaining and culturing embryonic stem cells
Pluripotent cells from the inner cell mass of the blastocyst
- Plated into culture dishes and grown in nutrient medium supplemented with serum, supported by an irradiated fibroblast feeder layer
- In vitro growth: Cells begin to divide over two weeks to form colonies. Colonies removed, dissociated and replated to allow further expansion. Cells continue to divide for several months without differentiation - allows expansion of pluripotent stem cell pool
State the source and methods of obtaining and culturing embryonic carcinoma cells
Derived from adult germ cell tumours - teratocarcinomas. Considered as malignant ECSs
Used as in vitro models
State the source and methods of obtaining and culturing Embryonic germ cell
Derived from primordial germ cells, the embryonic precursors of the gametes
Isolated from the embryonic gonad of post implantation embryo
Pluripotent differentiation capacity
State the source and methods of obtaining and culturing induced pluripotent stem cells
Adult stem cells transduced with small sets of genes (4 or fewer) that re-program the adult cells to give embryonic stem cell characteristics
State the source and methods of obtaining and culturing adult stem cells
Undifferentiated cells found in a variety of different adult tissues
ASCs can be isolated and expanded
Can be induced into different lineages
Describe Magnetic cell sorting (MACS)
Obtain mononuclear fraction from whole blood. Add magnetically labelled antibody to bind to CD34. Push cells through magnetic column - marked cells will stick to the magnet
Describe Fluorescent antibody cell sorting (FACS)
Used for separating small numbers of rare cells. Requires two types of cell marker
Obtain mononuclear fraction
Give an overview and describe the process of electrospinning
-Method of producing nano- and micro-scale fibrous networks, equivalent to ECM dimensions. Can be used to produce elongated or tubular structures
- Electrospinning creates mats of non-woven fibres
- Fibres can be nano-scale
- Matrix architecture can mimic extracellular matrix
- Nanofibrous materials promote cell:cell interactions
Electrical field applied to draw out a viscoelastic solution into a fine fibre.
Polymers (natural and synthetic) subjected to high voltage (several 1000 volts relative to grounded collecting plate).
Rotating collecting mechanism to align fibres
Give an application of polymers and microspinning
1) Vasular graft produced from polycaprolactone by electrospinning and functionalised with RGD. Used for a carotid artery transplant (rabbit).
Describe the concept of Solid Fabrication (SFF)
- 3D objects with complex geometries and internal architectures are produced in a layer-by-layer manner using data directly from a computer-generated model.
- Customised objects (scaffolds) can be produced using data generated by computed tomography (CT), magnetic resonance imaging (MRI) or designed de novo to fit purpose.
- Scan/design data is processed by computer aided design/computer-aided manufacturing (CAD/CAM) systems into a series of thin cross-sectional layers, which are bonded together.
Name and describe five different forms of solid freeform fabrication
- Three dimensional printing (3DP): Uses conventional inkjet technology to bind thin layers of powdered material together. Material is rolled over a platform that is sequentially lowered for each layer
- Fused deposition modelling (FDM): Stock filament material melted inside a moving heated nozzle. laid down as parallel lines. Direcion and spacing can be altered between consecutive layers, directed by x-y-z stage. Cells can not be encapsulated due to the heating process
- Stereolithography (SLA): UV beam selectively polmerises a liquid photocurable monomer. Beam is guided over the surface of a vat of liquid monomer, solidifying the first layer. Only a limited number of photopolymerisable materials
- Selective laser sintering (SLS): Laser slectively sinters (thermally bonds) particles of powdered material into thin layers. Material is rolled over a platform that is sequentially lowered for each layer
3d Bioplotter: Plots polymers (with or without cells) with defined 3D geometrics into a plotting medium
Describe Electron-beam lithography
- Generates nanoscale patterns. Possible due to very small spot size of the electrons: the resolution in UV lithography is limited by the wavelength of light
- Beam of electrons is directed across a surface covered with a “resist” material (PMMA), sensitive to electrons
- PMMA cast is then filled with PDMS elastomer used in microfabrication. When set this is lifted off and coated with a dry (gecko) or wet/dry adhesive (Geckel)
Give organ/tissue specific considerations when tissue engineering
Mechanical load - magnitude, habitual Oxygen tension Movement/Contraction Metabolic needs/activity Excitable
Describe the role of bioreactors
- Attempt to simulate in vivo conditions, with varying degrees of sophistication. Can proved environmental control, allow exchange of nutrients and provision of biological and mechanical cues
- Dynamic, perfused conditions become more important when considering in vitro growth and maintenence of 3D structures
Describe types of bioreactor
Spinner flasks - Cells can enter scaffold by convection. Stirring increases mass-transfer (movement of molecules from high to low concentrations)
Rotating Wall Vessel: Low shear stress, high mass transfer rates. Nasa developed to simulate microgravity
Hollow fibre: used to enhance mass-transfer to metabolically active cell types such as hepatocytes
Direct perfusion - medium flows directly through pores in the scaffold
Why is effective mass transfer important?
If, for example, the mass transfer of Oxygen from the bubbles in the fermentation broth is slow, then the rate of cell metabolism can become dependant on the rate of oxygen supply from the gas phase
Describe steps required to tissue engineering cardiac tissue
Call Supply: Undifferentiated HSCs induced using Activin A, BMP4 and Wnts to form beating cultures after 10-14 days
Biomimetic scaffold produced from poly glycerol sebacate (PGS) to improve perfusion of engineered cardiac tissue. Cardiomyoctes cultured in perfluorocarbon emulsion (oxygen carrier)
Electrical stimulation (cardiac-like) applied to cells to increase contraction amplitude. During this process cells go from being connected via gap junction to the assembly of striated myofibrils
Mechanical strain applied to cardiac cells
Describe an automated bioreactor
Biopsy taken from patient. Cells isolated, expanded, seeded on to scaffold and cultured within a closed system bioreactor monitored by researchers. Clinical records help provide input information and patient performance is measured to help time implantation
Give examples of the body as a bioreactor
1) Growth of cartilage the shape of an ear onthe back of a mouse
2) 56 year old man who had had tumour surgery to the mandible 8 years previously. (Had no lower jaw). Titanium mesh cage was filled with bone mineral blocks and 20ml bone marrow aspirate. BMP-7 added and then implanted into patients latissimus dorsi muscle for 7 weeks. New jaw then transplanted.
3) New zealand white rabbit study - created a bioreactor between surface of a tibia and the periosteum. Bioreactor space filled with calcium alginate gel (200 mm3). After 6 weeks, the neo-bone was harvested and transplanted into defect on contralateral limb. Radiographical and histological assessment of the neo-bone after six weeks showed that it had remodelled and integrated. (Concept also proven in human cadavers)
Decribe the possibilities for in situ tissue engineering
In situ TE does not necessarily involve generating new 3D tissue-like structures. May include (re)activation of resident (stem) cell populations and/or dedifferentation of somatic cells
e.g. Limb regeneration in Salamnders (dedifferentation of muscle, bone and connective tissue to form an undifferentiated mitotic blasterma. The blasterma re-patterns and re-differentaties into a limb
Deer antler regeneration - each spring deer shed antlers. A complex ‘blastema-like’ structure then forms (stem cell based, not dedifferentatiation)
Human kidney - De0differentation of resident cells - Recapitulating embryonic development to regrow the kidney in situ. Would require controlled dedifferentiation and may involve gene therapy. iPS cell model demonstrates that cell reprogramming is possible. Clinically attractive method
