Tissue Engineering

Culture of Cells for Tissue Engineering

Shu-Ping Lin, Ph.D.

Date: 02.21.2012

Institute of Biomedical Engineering E-mail: splin@dragon.nchu.edu.tw

Website: http://web.nchu.edu.tw/pweb/users/splin/

Introduction to Tissue Engineering and Basic Principles

of Cell Culture

Syllabus of Tissue Engineering Date Wk Topic

101/02/21 1 Introduction of Tissue Engineering and Basic Principles of Cell Culture

101/03/06 2 Tissue Engineering: Basic Considerations

101/03/13 3 Stem Cells for Tissue Engineering

101/03/20 4 Cellular Photoencapsulation in Hydrogels

101/03/27 5 Cartilage Tissue Engineering: Cell Sources, Lipid-Mediated Gene

Transfer, and Articular Cartilage/ Quiz 1

101/04/10 6 Ligament Tissue Engineering

101/04/17 7 Muscle Tissue Engineering: Human Skeletal Muscle for Clinical

Applications

101/04/24 8 Heart Tissue Engineering: Engineered Heart Tissue

101/05/01 9

Vessels Tissue Engineering: Tissue-Engineered Blood Vessels/ Quiz 2

101/05/08 10

Bone Tissue Engineering

101/05/15 11 Neuronal Tissue Engineering: Culture of Neuroendocrine and Neuronal

Cells for Tissue

101/05/22 12 Neuronal Tissue Engineering: Culture of Neuroendocrine and Neuronal

Cells for Tissue

101/05/29 13

Liver Tissue Engineering/ Quiz 3

101/06/05 14

Final Exam

101/06/12 15 Presentation of Paper Reading/ Final Report

Evaluation: Attendance 5％

Final Exam 45% Presentation 20% Quizzes 15% Final Report 15% Textbook: Culture of Cells for Tissue Engineering (Culture of Specialized Cells) By: Gordana Vunjak-Novakovic and R. Ian Freshney; Publisher: Wiley-Liss; 1 edition (February 3, 2006) Office Hours: Thur. 02:00 pm~04:00pm Phone:22840732 X302

TA: 江維元（BME）e-mail: boy20000_tw@hotmail.com

Research is presently being conducted on

several different types of tissues and organs,

including:

Cartilage

Ligament

Muscle

Heart

Blood Vessels

Bone

Neuronal

Liver

etc. (Skin, Kidney, ……)

Tissue Engineering

Tissue Engineering is the in vitro development (growth) of tissues or organs to replace or support the function of defective or injured body parts, or the directed management of the repair of tissues within the body (in vivo).

Tissue Engineering Interdisciplinary field that applies the principle of engineering and life

sciences to the development of biological substitutes that restore, maintain or augment tissue function

SJ Shieh and JP Vacanti Surgery 137 (2005) 1-7

An alternative to drug therapy, gene therapy and whole organ transplantation

Gene and drug therapy an option for treating the underlying disease if the molecular basis of the disease is understood

Less suitable for replacing the entire function of the cell

“Grow” organs in the lab

In-vitro tissue

culture

Biodegradable polymer scaffold

Osteoblasts

Chondrocytes

Hepatocytes

Enterocytes

Urothelial

Cells

Keratinocytes

Tissue Graft

Cartilage

Bone Liver

Intestine

Ureter

Skin

Tissue Organization

Before a tissue can be developed in vitro, first we must understand how tissues are organized. The basic tenet here is that:

“all tissues are comprised of

several levels of structural hierarchy”

These structural levels exist from the macroscopic level (centimeter range) all the way down the molecular level (nanometer range)

There can be as many as 7-10 distinct levels of structural organization in some tissues or organs

Hurdles of Tissue Engineering

Most successes have been limited to a vascular or thin tissues (< 200 mm)

• Skin, cartilage, cornea

The most important problems associated with thicker or more complex tissues include: The need for multiple cell types The need for the tissue to become vascularized

Vascularization of the 3-D construct is a critical and unresolved problem

Steps in Tissue Engineering Appropriate cell source must be identified, isolated and produced in sufficient numbers

Appropriate biocompatible material that can be used as a cell substrate or cell encapsulation material isolated or synthesized, manufactured into desired shape and dimensions

Cells seeded onto or into material, maintaining function, morphology

Engineered structure placed into appropriate in vivo site

Insoluble Matrix

Assemblies

CELLS

Cells

Soluble Matrix Molecules

Regulators of Matrix

Assembly

Matrix Bound Growth Factors

Bioactive Matrix

Soluble Growth Factors

Saints Cosmas and Damian performing a miraculous

transplantation

Oil painting on panel 168 x 133 cm., attributed to the Master of Los Balbases, Burgos, Spain, c. 1495

Reconstructive surgery: In 1970s the development of microsurgery allowed distant tissue transfer and reimplantation (vascular grafts and prosthetic articulation).

Tissue engineering does open radically new chapter in reconstructive medicine, for it is now deemed possible to reconstruct in the laboratory human living tissues and organs for in vivo, ex vivo, and even in vitro applications.

Tissue engineering is remarkably multidisciplinary, bringing together cell and molecular biologists, biochemists , engineers, pharmacologists, physicians, ..etc.

To obtain grafts for in vivo applications. The biological and mechanical functions are of utmost importance.

Biological functions: cell therapy

Mechanical functions: tissue templates.

The Aim of Tissue Engineering

Tissue-engineered substitutes are three-dimensional reconstructions that can be implanted into the human body, leading to rapid host implantation and acceptance.

The substitutes must have at least minimal biological and mechanical functions for such reparative role.

Historical Perspective

Tissue engineering has been considered one of the most influential new technologies for the future biomedicine.

The development of tissue engineering can be seen as heaving two phases:

1. The phase of exponential development and potential application. Is still continuing to evolve.

2. The phase brought about a flury of discoveries about stem cells.

Stem cells: S.c. had been known for many years.

Embrionic S.c.

Adult S.c. were found to be much more ubiquitous and to have more lineage plasticity than previously thought. (since 1998)

Embryonic stem cells, which are isolated from the inner cell mass of blastocysts

Adult stem cells, which are found in various tissues.

Potency Definitions Potency specifies the differentiation potential (the potential to differentiate into

different cell types) of the stem cell.

Totipotent (a.k.a omnipotent) stem cells can differentiate into embryonic and extraembryonic cell types. Such cells can construct a complete, viable organism. These cells are produced from the fusion of an egg and sperm cell. Cells produced by the first few divisions of the fertilized egg are also totipotent.

Pluripotent stem cells are the descendants of totipotent cells and can differentiate into nearly all cells, i.e. cells derived from any of the three germ layers.

Multipotent stem cells can differentiate into a number of cells, but only those of a closely related family of cells.

Oligopotent stem cells can differentiate into only a few cells, such as lymphoid or myeloid stem cells.

Unipotent cells can produce only one cell type, their own, but have the property of self-renewal, which distinguishes them from non-stem cells (e.g., muscle stem cells).

http://en.wikipedia.org/wiki/Stem_cell

Types of Culture Primary explantation versus disaggregation

Proliferation versus differentiation

Organotypic culture

By S. Waldman/B. Amsden

Culturing of Cells

Cell Culture

monolayer (adherent cells)

suspension (non-adherent cells)

three-dimensional (scaffolds or templates)

By S. Waldman/B. Amsden

Culturing of Cells

Sterilization Methods

ultra-violet light, 70% ethanol, steam autoclave, gamma irradiation, ethylene oxide gas

Growth Conditions

simulate physiological environment

pH 7.4, 37°C, 5% CO2, 95% relative humidity

culture (growth) media replenished periodically

Culture (Growth) Media

appropriate chemical environment

pH, osmolality, ionic strength, buffering agents

appropriate nutritional environment

nutrients, amino acids, vitamins, minerals, growth factors, etc.

Functional Subunits

The smallest level at which the basic function of the tissue/organ is provided is called a “functional subunit”:

functional subunits are in the order of ~100 mm (whereas cells are of the order of ~10 mm)

each organ is comprised between 10-100 x 106 functional subunits

each functional subunit is comprised of a mixture of different cell types and extracellular matrix (ECM) molecules

Separation of the functional subunit into individual cohorts (i.e. cells and ECM) leads to a loss of tissue function. For this reason, this is the scale that tissue-engineering tries to reconstruct.

So, how can the functional subunit be built in vitro?

By S. Waldman/B. Amsden

Microenvironment

Since cells are entirely responsible for synthesizing tissue constituents and assembly of the functional subunit, much attention is paid to the microenvironment surrounding the cell(s) of interest.

The microenvironment, which can be very different depending on the type of cell, is typically characterized by the following:

Cellularity

Cellular Communications

Local Chemical Environment

Local Geometry

By S. Waldman/B. Amsden

Cellularity

Packing Density:

maximum theoretical packing density is about 1 x 109 cells/cm3

cell densities in tissues typically vary between 10 – 500 x 106 cells/cm3

relates to about 100 - 500 cells per microenvironment (100 mm)3

extreme cases, such as cartilage which has ~ 1 cell per (100 mm)3

thus its microenvironment is essentially 1 cell plus associated ECM

Cellular Communication:

Cells communicate in three principal ways:

secretion of soluble signals

cell-to-cell contact

cell-ECM interactions

Cellular communication can affect all “cellular fate” processes (migration, replication, differentiation, apoptosis) and the method(s) of communication used depends, in part, on how the cells are packed within the tissue.

By S. Waldman/B. Amsden

Cellular Communications

Soluble Signals:

includes small proteins such as growth factors and cytokines (15-20 kDa), steroids, hormones

bind to membrane receptors usually with high affinity (low binding constants: 10-100 pM)

By S. Waldman/B. Amsden

Cellular Communications

Cell-to-Cell Contact:

some membrane receptors are adhesive molecules

adherent junctions and desmosomes

other serve to create junctions between adjacent cells allowing for direct cytoplasmic communication

gap junctions

1.5-2 nm diameter and only allow transport of small molecules ~1 kDa

By S. Waldman/B. Amsden

Cellular Communications

Cell-ECM Interactions:

ECM is multifunctional and also provides a substrate that cells can communicate through

since cells synthesize the ECM, they can modify the ECM to elicit specific cellular responses

cells possess several specialized receptors that allow for cell-ECM interactions

integrins, CD44, etc.

also a mechanism by with cells respond to external stimuli (“mechanical transducers”)

By S. Waldman/B. Amsden

Chemical Environment

Oxygenation:

mammalian cells do not consume oxygen rapidly but uptake is large in comparison to the amount in blood or culture media

air-saturated aqueous media (37°C) contains only 21 mM O2

mammalian cells consume O2 at rate of 0.05-0.5 mmol/106 cells/hour

cell cultures for tissue engineering have relatively large cell densities (106 cells/mL) which results in total O2 depletion in 0.4-4 hours!

concentration must be within a specific range since oxygenation affects a variety of physiological functions

low O2 concentration can retard growth

high O2 concentration can be inhibitory or toxic (oxidative stress)

Metabolism:

typically, there are no transport limitations for major nutrients although uptake rate depends on their local concentrations

glucose uptake rate: 0.1-0.5 mmol/106 cells/hour

amino acid uptake rate: 1.0-5.0 nmol/106 cells/hour

By S. Waldman/B. Amsden

Local Geometry

Geometry of the microenvironment depends on the individual tissue:

needs to be re-created for proper tissue growth

two-dimensional layers or sheets

three-dimensional arrangements

transport issues

local geometry also affects how cells interact with the ECM

remember, the ECM serves as a substrate for cellular communications

For these reasons, considerable effort has been geared at creating artificial ECM’s (aka scaffolds) to provide the appropriate substrate to guide in vitro tissue growth and development.

By S. Waldman/B. Amsden

Tissue Engineering Scaffolds

Scaffold Materials:

synthetic polymers

poly(lactide) ,poly(lactide-co-glycolide), poly(caprolactone)….

foams, hydrogels, fibres, thin films

natural polymers

collagen, elastin, fibrin, chitosan, alginate….

fibres, hydrogels

ceramic

calcium phosphate based for bone tissue engineering

porous structures

permanent versus resorbable

degradation typically by hydrolysis (except for natural materials)

must match degradation rate with tissue growth

Chemical and Physical Modifications (synthetic materials):

attachment of growth factors, binding sites for integrins, etc.

nanoscale physical features

By S. Waldman/B. Amsden

Tissue Engineering Scaffolds

smooth muscle cells on unmodified poly(CL-LA) elastomer (L)

and modified surface having bound peptide sequence (R)

By S. Waldman/B. Amsden

Cell Sources

Since the ultimate goal of tissue engineering is to develop replacement tissue (or organs) for individuals, the use of autologous cells would avoid any potential immunological complications.

Various classifications of cells used in tissue engineering applications:

primary cells

differentiated cells harvested from the patient (tissue biopsy)

low cellular yield (can only harvest so much)

potential age-related problems

passaged cells

serial expansion of primary cells (can increase population by 100-1000X)

tendency to either lose potency or de-differentiate with too many passages

stem cells

undifferentiated cells

self-renewal capability (unlimited?)

can differentiate into functional cell types

very rare

By S. Waldman/B. Amsden

Stem Cells

Stem cells naturally exist in essentially all tissues (especially those that rapidly proliferate or remodel) and are present in the circulation.

There are two predominant lineages of stem cells:

mesenchymal

give rise to connective tissues (bone, cartilage, etc.)

although found in some tissues, typically isolated from bone marrow

hematopoietic

give rise to blood cells and lymphocytes

isolated from bone marrow, blood (umbilical cord)

Stem cells are rare; bone marrow typically has:

a single mesenchymal stem cell for every 1,000,000 myeloid cells

a single hematopoietic stem cell for every 100,000 myeloid cells

By S. Waldman/B. Amsden

Stem Cells (Mesenchymal)

By S. Waldman/B. Amsden

Stem Cells (Hematopoietic)

By S. Waldman/B. Amsden

Proliferation versus Commitment

Proliferation

Clonal

Succession

Deterministic

or Stochastic

Succession

Stem Cell

Commitment or Differentiation

By S. Waldman/B. Amsden

Stem Cells Identification

Stem cells are identified by the expression of specific antigens on their surface, for example:

hematopoietic stem cells express CD45, CD34 and CD14

mesenchymal stem cells do not express these markers (i.e. CD34-, CD45-, CD14-)

Selective separation of positive marker cells (in a mixed cell population) can be done by several techniques (e.g. immunomagnetic methods).

Characterization and Commitment

The most common approach to characterize multi-lineage- or single lineage-committed stem cells is through colony-forming assays:

cells grown under culture conditions that promote their proliferation and differentiation

the clonal progeny of a single progenitor cell stay together to form a new colony of mature cells

By S. Waldman/B. Amsden

colony-forming assays are used to:

characterize stem cells from different sources (e.g. BM, umbilical cord blood)

investigate responses to growth factors, cytokines and other drugs

expansion, commitment, etc.

quality control for collection, processing and cryopreservation

Colony-Forming Units (CFUs)