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CLINICAL PATHOLOGY > Primary Cell Culture > Flashcards

Primary Cell Culture Flashcards

1
Q

What is a primary cell culture?

A
  • Technique by which cells from primary tissues or cell suspensions are grown under controlled conditions in vitro
  • Cells will divide and or differentiate like normal cells, carrying out their normal function
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2
Q

Provide some examples of non-haemopoietic primary cultures

A

Liver, Muscle, Skin, Nerves, Fibroblasts, Endothelial

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3
Q

Provide some examples of haemopoietic primary cultures

A
  • Stem, Progenitor cells
  • T and B cells
  • Monocytes, Macrophages
  • Osteoblasts
  • Dendritic cells
  • Neutrophils
    • Eosinophils, Basophils, Mast cells
  • Erythrocytes
  • Megakaryocytes
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4
Q

What can primary cultures be established from?

A
  • Haemopoetic cells
    • Giving rise to mature blood cell types
  • Non-haematopoietic cells
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5
Q

What are the differences between cell cultures and cell lines?

A
  • Primary cell cultures are directly derived from tissues
    • Cell lines are transformed cells which may be genetically identical
  • Interpatient variabillity
    • In a cell line every cell is identical
  • Finite lifespan (cant consistently maintain O2 and growth medium)
    • A cell line will divide and reproduce itself exactly the same
  • Cells divide and/or differentiate
    • A cell line will divide and reproduce itself exactly the same
  • Cells carry out normal funciton
    • Because cell lines are transformed it may not have a normal function
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6
Q

By what process do we manipulate tissues to get a single cell suspension for primary cell cultures?

A

Disaggregation

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7
Q

List some disaggregation techniques

A
  • Cells migrate out of explant
  • Mechanical dissociation (mincing, sieving, pipetting)
  • Enzymatic dissociation (trypsin, collagenase, hylaluronidase)
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8
Q

What cell does not need to be disaggregated?

A

Haematopoietic cells do not need to be disaggregated as they are already in single cell suspension

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9
Q

What are sources of stem cells?

A
  • Bone marrow aspirate
  • Umbilical cord blood
  • Mobilised peripheral blood
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10
Q

Where does haematopoiesis occur in children?

A

Red bone marrow

Liver

Spleen

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11
Q

Where does haematopoeisis occur in adults?

A

In adults (after 20 years), the bone marrow retreats and haematopoiesis only occur in:

  • End of long bones (e.g femur, humerus)
  • Skull
  • Vertebrae
  • Sternum
  • Pelvis
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12
Q

What is lifecycle of haematopoetic cells?

A

Pluripotent stem cells → Early progenitors → Late Progenitors → Immature Precursors → Mature Blood Cells

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13
Q

Why are you not able to see the lineage of progenitor cells?

A
  • They are undifferentiated cells and therefore do not show any mature characteristics
    • HOWEVER they are already commited to a specific lineage and make a specific type of cell
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14
Q

Stem Cells

A

pluripotent, give rise to all lineages, self-renew, rare cells, responsible for engraftment

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15
Q

Progenitor cells

A

Undifferentiated, not distinguished by morphology, committed to one or more lineages, detected in colony forming assays

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16
Q

Precursor cells

A

immature but recognisable, cells starting to differentiate, dew final divisions before forming mature cells

17
Q

What is haemopoiesis?

A

process whereby immature precursor cells develop into mature blood cells

18
Q

How do haematopoietic growth factors?

A
  1. Bind to cell surface transmembrane receptors
  2. Stimulate growth + survival of progenitors
19
Q

What are stromal cells?

Provide some examples of stromal cells.

A

Stromal cells are cells in the bone marrow which form the environment for stem cells

  • Fibroblasts, Macrophages, Endothelial cells, adipocytes
20
Q

What do stromal cells produce on their surface?

A

Produce ECM (extracellular matrix) proteins on their surface:

  • Collagen I, II, III, IV
  • Laminin
  • Fibronectin
  • Hemonectin
  • Thrombospondin
  • Proteoglycans
21
Q

What do stromal cells contain on their surface?

A

Adhesion receptors

  • Integrins
  • Selectin
  • CD44
  • Lectins
22
Q

What cells will produce cytokines and inhibitors?

A

Stromal cells

23
Q

How are stem cells in interaction with stromal cells?

A

The stem cells will have equivalent receptors to adhesion receptors which will recognise cytokines and inhibitors produced by stromal cells.

24
Q

What are different techniques to identify the haematopoesis stages that are not cell culture?

A
  • Phenotype
  • Fluorescent stains/ cytotoxic drugs
  • Assays
25
Q

Phenotypic techniques to identify haematopoetic cells

A
  • Stem cells and progenitor cells (early+ late) are all posotive for CD34, mature blood cells are negative CD34
  • A stem cells are Lin negative, mature cells are Lin positive
26
Q

How do fluorescent stains/ cytotoxic drug techniques work to identify haematopoetic cells?

A
  • Rh123 is a fluorescent dye = stains mitochondria. Stem cells out of the cycle will not stain for that. Actively cycling cells will stain bright
  • 5-FU is a cytotoxic drug and will only affect cycling cells. Cells out of cycle wil be resistant to this drug. Adding this drug will kill cycling cells
27
Q

Assay techniques to identify haematopoietic cells

A
  • Some assays can recognise and grow early stem cells
  • Colony forming unit assays for lineage commited progenitors
28
Q

How to we undertake cell processing?

A
  • Haematopoietic stem cells do not require processing as they are already in a single cell suspension
  • There are various techniques which will enrich and purify stem cells ready for culture
    • Erythrocyte lysis
      • Enrichment of stem cells
    • Density gradient centrifugation
      • This will remove some cells and enriches for others
    • Adherance depletion
      • Bone marrow on a sticking culture, remove non-adherant cells through pipetting
    • Antibody depletion
      • Can use antibodies for Lin antigens, these will recognise mature cells and deplete
    • Antibody selection
      • Can use antibodies for stem cells and select them via flow cytometry/ magnetic beads
      • Label bone marrow with CD34 (e.g)
29
Q

What is a colony assay?

A
  • This is where progenitors will grow to form colonies of mature cells
  • Progenitors are also called CFUs (colony forming units)
  • Progenitors will grow from 30 to several hundreds and thousands
30
Q

How is a colony forming assay formed?

A
  1. Take single cell suspension and grow it with growth factors in a semi solid medium (agar-methylcellulose) in an incubator for 7-14 days
  2. The progenitor cells will replicate to produce a colony of more mature cells (most one lineage only )
  3. This can be seen down a microscope and can be identified as cell types
31
Q

What are some applications of primary cell cultures?

A

Research - basic haematopoeisis and carcinogenesis

Testing for toxicity of chemotherapeutic agents and carcinogens

Generate cells for stem cells transplantation and manipulation

CLINICAL PATHOLOGY (52 decks)

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