動物細胞培養 授課老師 : 顏嘉宏 助理教授 教科書 : Culture of Animal Cells- a manual of basic technique (5 th Edition, Freshney R.I.)

Tissue Culture 1.Cell culture: a culture derived from dispersed cells 2.Organ culture: a three-dimensional culture of undisaggregated tissue retaining some of the histological features of the tissue 3.Histotypic culture: cells have been grown to re-create a 3-D structure with tissuelike cell density 4.Organotypic culture: recombining cells of different lineages

Figure 25-2 Organotypic culture Histotypic culture “ one cell type ” [more than one layer] “ two cell type ”

Tissue Culture Application (I) (1) (2) (3) (4) (5) (6) (7) (8) (9)

Tissue Culture Application (II) 1. Production of antiviral vaccines 2.Understanding of neoplasia (caner research) 3.Transfer of DNA to the cultured cells (or siRNA) 4.Monoclonal antibody production (immunology) 5.Production of human growth hormone, insulin, interferon 6.Stem cell culture differentiate into neurons 7.Implanting normal fetal neurons into patients with Parkinson diseases 8.Homografting and reconstructive surgery using individual ’ s own cells (tissue engineering) 9.In vitro fertilization (embryo culture)

Hybridoma Fig [P3.653 Myeloma] (HAT selection: medium containing hypoxanthine, aminopterin, thymidine) (B cells+ other cells)

(1) (2) (O 2 & CO 2 ) Poorly defined materials: serum, supplementations, matrix, … Table 1-2

Cell line homogeneity “ After one or two passages, cultured cell lines assume a homogeneous constitution, as the cells are randomly mixed at each transfer and the selective pressure of the culture condition tends to produce a homogeneous culture of the most vigorous cell type ” ?

Reagent saving “ Less reagent is required than for injection in vivo, where 90% is lost by excretion and distribution to tissues other than the interested cells under study ” In Vitro Dosage v.s. In Vivo Dosage ?

Reduction of animal use “ In vitro modeling of in vivo conditions ”

(Table 13-2) Table 1-3

Microbial Contamination “ Animal cells grow much less rapidly than contaminants (bacteria, molds, yeasts) ” Cross-Contamination “ Many cell lines in common use are not what they are claimed to be, but have been cross-contaminated with HeLa or other growing cell line ”

Cross-Contamination(I)

Cross-Contamination(II)

Henrietta Lacks Cross-Contaminated cell lines ” HeLa cell ” George Otto Gey ” HeLa cell ” Cervical Cancer

Cost “ The cost of producing cells in culture is about 10 times that of using animal tissue ” “ Semimicro- or Micro- Assays ” : reduced manipulation time (Quicker !!)

Genetic and Phenotypic Instability “ Dedifferentitation: a process assumed to be the reversal of differentiation ” “ overgrowth of undifferentiated cells ” “ Loss of the phenotypic characteristics typical of the tissue ” (1)Specific cell interactions characteristics of the histology of the tissue are lost; (2)The culture environment lacks systemically homeostatic regulation systems (nervous and endocrine system) Possible Reasons:

Origin of cells “ If differentiated properties are lost, it is difficult to relate the cultured cells to functional cells ” “ Stable Expressed Markers ” ?

Types of tissue culture (I):

Types of tissue culture (II): (1)Organ culture: -- retention of a spherical or three-dimensional shape -- retention of specific histological interaction (2)Primary explant culture: -- a fragment of tissue -- after attachment, migration is promoted in the plane of the solid substrate (3)Cell culture: -- as an adherent monolayer on a solid substrate (various cell type) -- as a suspension in the culture medium (few cell type)

Table 1-4 culture

Cell Culture (I) 1.Cell proliferation is often found 2.Dispersed by enzymatic treatment or simple dilution into fresh vessels [Reseeded or Subcultured] 3.Parent cells --- ” passage ” > daughter cells (cell line) 4.Cell line ” selection ” ----> Subline (cell strain) 5.Adherent Monolayer culture: -- Anchorage dependence: attachment to the substrate is a prerequisite for cell proliferation -- Most normal cells 6.Suspension culture: --Anchorage independence: cells can survive and proliferate w/o attachment --Hematopoietic cells, transformed cells, cells from malignant tumors

Cell Culture (II) 7. The derivation of continuous cell line or cell strain usually implies a phenotypic change, or transformation 8. Cultured cell lines are more representative of precursor cells [most differentiated cells do not divide] 9. Cultured cells lack the potential for cell-cell and cell-matrix interaction

Table 1-5