Lecture 4: Cell, Tissue And Organ Culture

Question 1

Tissue engineering

Answer 1

• Simple - e.g. single cell type such as chondrocytes (secrete cartilage) & keratinocytes (found in skin) - mainly 2D
• Complex - e.g. Bone & liver - mainly 3D, some require vascular supply
• Organoids

Question 2

Skin

Answer 2

Epidermis: Mainly keratinocytes (prickle cells) & smaller numbers of melanocytes
Dermis: Connective tissue, collagen, fibroblasts (secrete collagen), blood vessels etc

Question 3

Skin functions

Answer 3

• protection
• immunological (barrier)
• fluid, protein, & electrolyte homeostasis
• thermoregulation
• neurosensory

Question 4

Skin grafts

Answer 4

Source of cells:
- cadavers or biopsy (e.g. neonatal foreskins) (allografts (from other people))
- porcine (xenografts/heterografts) e.g. permacol
- individual (autograft (your own cells) e.g. Epicel & Laserskin cultured epidermal autografts

Question 5

Types of skin grafts

Answer 5

• one layer - e.g. epidermal kerartinocytes - Epicel®
• two layers - e.g. both keratinocytes & fibroblasts - Apligraf®

Question 6

One layer

Question 7

Epicel®

Answer 7

• Epicel® cultured epidermal autografts (CEA) (from own individual) is an aseptically processed wound dressing comprised of the patients own (autologous) ….

Question 8

Epicel®

Answer 8

• is indicated for use in patients who have deep dermal or full thickness burns comprising a total body surface area of greater than or equal to 30%

Question 9

Two layers

Answer 9

• equivalent of a epidermis & dermis

Question 10

Laser skin

Question 11

.

Question 12

.

Question 13

COMPLEX: Bone

Answer 13

• skeletal stem cells (proliferative ability)
• require scaffolds

Question 14

What are scaffolds?

Answer 14

• porous & degradable mesh-works that cells attach to
• recreate ECM in terms of physical & chemical properties. Provided growth factors & allow cellular proliferation
  …

Question 15

Scaffolds

Question 16

Examples of advances in tissue engineering: BONE

Answer 16

Compact bone structure:
- central blood vessel
- cell extensions

Question 17

Bone

Answer 17

cellular components (ca. 15% of bone)
- Osteoblasts - cells that secrete the matrix
- Osteocytes - cells that are formed from osteoblasts & may have limited powers of matrix formation
- Osteoclasts - cells that digest & re-model bone (a type of macrophage)

Matrix:
- osteoid collagen - a type of I collagen
- proteoglycan
- GAG
…..

Question 18

‘Printing’ a bone scaffold

Question 19

Producing osteoblasts from induced pluripotent stem cells

Answer 19

• addition of specific growth factors
• addition of calcium phosphate
• osteogenic growth medium

Question 20

Exogenous adenosine induced osteogenic differentiation of human induced pluripotent stem cells (hiPSCs)

Question 21

In vitro bone-forming ability of hiPSC-derived cells

Question 22

hiPSC-derived osteoblasts (Ad-hiPSCs) contribute to the healing of critical-
sized bone defects through the formation of vascularized neobone tissue

Question 23

Examples of advances in tissue engineering: EYE

Answer 23

The human eye

Question 24

Cornea (outer layer)

Answer 24

epithelium (outer layer): stratified, squamous epithelium
Bowman’s layer: collagen
Stroma: collagen plus keratinocytes
Endothelium: cuboidal

Question 25

Corneal implant made from recombinant human collagen type III

Question 26

Cross section of normal vs Biosynthetic vs Human donor

Question 27

Diseases affecting the retina (back of the eye)

Answer 27

- age related macular degeneration (ARMD; photoreceptor loss) - Glaucoma - Retinis pigmentosa …..

Question 28

ARMD

Answer 28

Two types: 1. **Dry ARMD** - slow to develop, caused by waste product deposits (drusen) that build up in the macula

Question 29

Symptoms of dry ARMD

Question 30

ARMD

Answer 30

Two types: 2. **Wet ARMD**: rapid onset, more serious, caused by blood vessel rupture

Question 31

iPSCs and age related macular degeneration (ARMD)

Answer 31

- wet AMD involves damage to retinal pigment epithelial cells - these cells are in a monolayer that supports the overlying photoreceptors & underlying choroidal vasculature in the eye - study in 2017 … took skin fibroblasts from 77 year old patient with AMD ….

Question 32

How stem cells may cure blindness

Question 33

Advances in retinal developemnt in vitro

Answer 33

Day 0: culture embryonic stem cells with a dash of growth factors Add Matrigel, a cell substrate Day 2 - 4: cells form embryoid bodies; many become neural precursor cells

Question 34

.

Question 35

Examples of advances in tissue engineering: VASCUALR GRAFTS