Cell and Tissue Engineering

Question 1

Give a short timeline of the development of regenerative medicine

Answer 1

1960’s - first adult stem cell (mouse) isolated
1981- Mouse embryonic stem cells isolated
1998 - Human ESCs isolated
2007- Human iPSCs cultivated

Question 2

Give reasons for why regenerative medicine is required

Answer 2

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

Question 3

Give examples of current therapies

Answer 3

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

Question 4

Define Tissue egineering

Answer 4

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

Question 5

Define Cell Engineering

Answer 5

The process of modifying cells for therapeutic application. May involve genetic, mechanical, chemical modification and reprogramming

Question 6

State the source and methods of obtaining and culturing primary cells

Answer 6

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

Question 7

State the source and methods of obtaining and culturing embryonic stem cells

Answer 7

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

Question 8

State the source and methods of obtaining and culturing embryonic carcinoma cells

Answer 8

Derived from adult germ cell tumours - teratocarcinomas. Considered as malignant ECSs
Used as in vitro models

Question 9

State the source and methods of obtaining and culturing Embryonic germ cell

Answer 9

Derived from primordial germ cells, the embryonic precursors of the gametes
Isolated from the embryonic gonad of post implantation embryo
Pluripotent differentiation capacity

Question 10

State the source and methods of obtaining and culturing induced pluripotent stem cells

Answer 10

Adult stem cells transduced with small sets of genes (4 or fewer) that re-program the adult cells to give embryonic stem cell characteristics

Question 11

State the source and methods of obtaining and culturing adult stem cells

Answer 11

Undifferentiated cells found in a variety of different adult tissues

ASCs can be isolated and expanded

Can be induced into different lineages

Question 12

Describe Magnetic cell sorting (MACS)

Answer 12

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

Question 13

Describe Fluorescent antibody cell sorting (FACS)

Answer 13

Used for separating small numbers of rare cells. Requires two types of cell marker

Obtain mononuclear fraction

Question 14

Give an overview and describe the process of electrospinning

Answer 14

-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

Question 15

Give an application of polymers and microspinning

Answer 15

1) Vasular graft produced from polycaprolactone by electrospinning and functionalised with RGD. Used for a carotid artery transplant (rabbit).

Question 16

Describe the concept of Solid Fabrication (SFF)

Answer 16

• 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.

Question 17

Name and describe five different forms of solid freeform fabrication

Answer 17

• 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

Question 18

Describe Electron-beam lithography

Answer 18

• 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)

Question 19

Give organ/tissue specific considerations when tissue engineering

Answer 19

Mechanical load - magnitude, habitual
Oxygen tension
Movement/Contraction
Metabolic needs/activity
Excitable

Question 20

Describe the role of bioreactors

Answer 20

• 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

Question 21

Describe types of bioreactor

Answer 21

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

Question 22

Why is effective mass transfer important?

Answer 22

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

Question 23

Describe steps required to tissue engineering cardiac tissue

Answer 23

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

Question 24

Describe an automated bioreactor

Answer 24

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

Question 25

Give examples of the body as a bioreactor

Answer 25

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)

Question 26

Decribe the possibilities for in situ tissue engineering

Answer 26

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