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Regenerative Medicine > Analytical Cytometry > Flashcards

Analytical Cytometry Flashcards

1
Q

What are inline measurements?

A

Measurements performed in an automated manner inside the bioreactor

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2
Q

What are are offline measurements?

A

Measurements performed manually. They require sampling

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3
Q

What’s a sensor in terms of bioprocessing?

A

A device that detects and responds to some type of input from the physical environment. The specific input could be light, heat, motion, moisture, pressure and etc.

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4
Q

What are fluorophores?

A

A fluorescent chemical compound that can re emit light upon light excitation

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5
Q

What’s flow cytometry?

A

A technique used to detect and measure physical and chemical characteristics of a population of cells or particles.

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6
Q

What are the 4 phases of the cell growth cycle and where do they happen in terms of high or low cell count vs time?

A
  1. Lag phase - low cell count low time
  2. Exponential (log) phase - increase by a steep upward curve from lag phase
  3. Stationary phase - highest cell count in cycle at midway on time axis increase from exponential phase. There’s a period of stationary cell count
  4. Death phase - decrease from stationary phase by approximately the same amount of increase from exponential to stationary . Straight line
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7
Q

Why is it important to track the biomass?

A

Optimise the culture
- induce systems at best time
- calculate accurate feed rate times
- harvest the product at the correct time

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8
Q

What properties can be measured when looking at the cytology of bacterial cells?

A

Glucose, ph, do2 -> process control (sensor)

Total cell count

Optical density

Dry cell weight

Viable cell count - CFU,methylene blue

Others:
Metabolomics
Genomics
Proteomics
Transcriptomics

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9
Q

What is optical density used to measure?

A

Measure only of proliferation

Rarely takes into account the medium composition or changes to medium composition

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10
Q

What does dry cell weight measure and how long till results are available

A

Measure only of proliferation

Results available at least 12h later

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11
Q

What’s specific gravity measurement for?

A

Relative measure of how a process is proceeding

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12
Q

What is measured when looking at the cytology for mammalian cells?

A

Glucose and metabolites (lactate, ammonia)
Ph, do2
Total cell count
Viable cell count - using strains such as trypan blue,DAPI, propidium iodide which penetrate damaged cell membranes

Others:
Metabolomics
Genomics
Proteomics
Transcriptomics

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13
Q

What are omic technologies?

A

Universal detection of
Proteomics
Metabolomics
Genomics
Transcriptomics - DNA , mRNA, microarrays

Assume homogeneity
Huge amounts of data

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14
Q

Explain manual cell counting

A

Haemocytometer staining and trypan blue staining

Trypan blue is negatively charged due that only stains cells with compromised cell membrane so indicates cell death

Dead cells are stained blue

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15
Q

Explain automated cell counting

A

Nucleocounter
- uses either multiple chamber slides or via-1 cassettes
- staining with acridine orange (live cells) and DAPI (damages membranes)

Acridine orange is an organic compound used as nucleic acid-selective fluorescent cationic dye useful for cell cycle determination. Being cell permeable, it interacted with DNA and RNA by intercalation or electrostatic attractions, respectively

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16
Q

What does automated cell counting rely on and assume?

A

Relies on software estimation
Assumes homogeneity

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17
Q

What are some indirect methods of monitoring cell growth and viability?

A

Indirect methods
- luminescent atp monitoring
- fluorescent proliferation assays: presto blue, alamar blue
- colorimetric proliferation assays: XTT, MTT

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18
Q

How does calcein work

A

virtually non fluorescent and once it enters live cells under effect of intracellular enzymes, is converted to polyanionic dye calcein that is well retained within living cells

Producing an intense green fluorescence

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19
Q

Explain what ethidium homodimer does?

A

Enters cells with damaged membranes and undergoes 40 fold enhancement of fluorescence upon binding to nucleic acids

producing a bright red fluorescence in dead cells

20
Q

Explain the process of proliferation assays and give a disadvantage

A
  1. Grow cells in plate wells
  2. Add prestoBlue reagent and incubate for 10 mins
  3. Measure fluorescence (red)
  4. Measure fluorescence (green)

Requires precise control and is time-consuming

Can use either presto blue or almar blue

Requires calibration c

21
Q

List the types of assays and their properties

A

PrestoBlue - resazurin based - single addition - >=10mins - absorbable or fluorescence - live - is compatible with phenol red - 12 cells per well

AlmarBlue - resazurin based - single addition - 1-4 hours - absorbable and fluorescence- live - is compatible - 50 cells/wells

Resazurin powder- resazurin based - weigh powder,prepare solution + single addition -4h - absorbable or fluorescence- live cells - is compatible 100 cells/well

MTT powder - tetrazolium based - weigh powder, prepare solution, + 2 additions, absorbance, cell format is lysis/endpoint, not compatible with phenol red, 1000 cells/well

XTT powder - tertrazolium based, weigh powder, prepare solution, + 2 additions, 2-4 hours, absorbance, lysis/endpoint, compatible, 1000 cells/well

Equipment used - spectrometer, photometer, micro plate reader

22
Q

What aspects of cell metabolism can be monitored and why?

A

Glucose
Glutamine
Lactate
Ammonia
Glutamate
Lactate dehydrogenase (LDH)

This monitoring provides insight to creating an a optimised informed feeding regime

23
Q

How can we measure the cell metabolites?

A

Using bioanalysers

Cedex HT

Nova biomedical

24
Q

What is microscopy?

A

Magnifying cells to view them,
Optical / light microscopy
- bright field
- phase contrast
- DIC

Fluorescence microscopy

25
Q

Explain a optical/light microscope

A

Uses photons (light)
Living or fixed sample
3D
Quick
Inexpensive
Up to 1500x magnification
Contrast techniques-> bright field, phase contrast, DIC
options for complex analysis -> time lapse and z stacking

26
Q

What does DIC stand for?

A

Differential interference contrast

27
Q

What is time lapse microscopy used for?

A

Observing live cells like culture or tissue samples

Growing life forms

Can use a CELL IQ or a bio station (Nikon)

^ these are incubators with built in microscopes can record over set periods of time

Can be used for differentiation

Only works for adherent cells only 2D

28
Q

What is fluorescence microscopy?

A

Inverted microscope that uses fluorescence

Surrounded by a temperature control chamber

Fluoresces different colours red, green, blue and yellow

Ideal for live cell imaging and time lapsing

29
Q

What are fluorophores?

A

Fluorophores absorb energy (short wavelength) and emit at a longer wavelength (strokes shift)

Filters let us see the individual light from each dye

We can attach fluorophores to molecules/areas of interest

30
Q

Name 2 fluorophores

A

Dylight 405

Alexa fluor 405

Pacific blue

FITC

31
Q

What is immunofluorescemce staining?

A

Technique to assess expression of specific cell markers (intracellular or surface)

Utilises antibodies and fluorophores

Imaging is done on a fluorescence microscope

32
Q

What’s direct and indirect labelling when concerned with immunofluorescemce staining?

A

Direct labelling
- attacking labelling antibodies directly to the cell receptors
- usually used MAb for specificity

Indirect labelling
- attaching labels to antibodies that attach to cell receptors
- 2 steps -> primary and secondary antibodies
- primary - usually MAb for specificity
Secondary - usually PAb for increased sensitivity. Carries the fluoroscope

33
Q

Name a few benefits of microscopy

A

Provides morphological data

Can be used for ultrastructure
1) electron microscopy
2) immuno-gold labelling

34
Q

Give the limitations of microscopy

A

Assumption of homogeneity (population vs single cell measurement)

Concerns with methods themselves
- viable, but not culturable cells
- false positives - need to understand the technique and any pitfalls
- human input
- time consuming, offline

Ideal analytical methods
- online or in lines
- rapid
- high throughout
- automated
- help you control your process

35
Q

What’s kind of sensors and probes can be found in a bioreactor?

A

Standard probes :
- PH probe
- Temperature probe
- DO (dissolved oxygen) probes

36
Q

What specialised probes are used for cell cultures?

A

Biomass probes
- measure Optical Density
- measure impedance

37
Q

What do the Hamilton incyte and dencytee probes measure?

A

Incyte
- measures permitivity of viable cells
- the signal can be correlated to the viable cell density measured offline
- can be used directly in the bioreactor for real time process control, eliminating the need for offline sampling

Dencytee
- total cell density measurement
- based in optical density or the turbidity of a suspension at NIR wavelength

38
Q

What does the futura sensor do ?

A

Measures the impedance of variable cells at radio frequencies

it involves applying an electrical field to polarise cells

The signal can be correlated to the viable cell density or biomass

Can be used directly in the bioreactor for real time process control, eliminating the need for offline sampling

39
Q

How does measuring the impedance work?

A

When an electrical field is applied to cells in an aqueous, ionic solution the ions in the solution are forced to move

Ions both inside and outside the cells move until they encounter the plasma membranes, which will act as insulating physical barriers to prevent further movement

Bulk movement causes a charge separation or polarisation of the cells

The magnitude of resulting field induced separations is measured by capacitance

As the volume fraction of cells in a suspension increases, the number of polarised membranes also increases to yield a higher measured capacitance

Dead cells and non biomass solids have no intact plasma membranes and so do not polarise significantly, nor do they contribute significantly to the capacitance of a cell suspension

40
Q

What’s flow cytometry?

A

A technique used to detect and measure physical and chemical characteristics of a population of cells or particles. A sample containing cells or particles is suspended in a fluid injected into the flow cytometer instrument

Allows large populations to be studied

Small changes can be significant

More quantitative than microscopy

Quicker and more consistent

41
Q

What are the advantages of flow cytometry?

A

Highly versatile

Highly sensitive

Real time, statistically reliable information

Multiple parameter can be measured

Cell number

Cell structure

Protein expression

Cellular metabolic activity -> cell physiology and viability

Individual cell level

Allows informed decision about a process to be made

42
Q

What are the various variations of flow cytometry specifications?

A

Can be single sample or high throughput

Multiple lasers can be 3,4,5

Detection of up to 16 colours

Particle size microns to nano meters

With it without cell sorting

43
Q

What is forward and side scatter?

A

Cells or particle passing through the beam scatter light which is detected as forward scatter and side scatter

Forward scatter correlates with cell size and as is proportional to the granularity of the cells

Cell population can often distinguish based on differences in their size and granularity alone

44
Q

Considering blood sample as an example explain how Yh front scatter and back scatter would behave

A

Larger and more granular granulocyte cells produce a large population with high side scatter and front scatter

Monocytes are large cells but not so granular so these produce a separate population with higher front scatter and lower side scatter

Smaller lymphocytes and lymphoblasts produce a separate population with less front scatter. They are not granular cells so have low side scatter

45
Q

What are the two types of fluorophores?

A

FITC (ex/Em) : 495/519nm - green

PE (ex/Em) : 496/578 nm - yellow/orange

46
Q

How does flow cytometry work?

A

As the fluorescing cells passes through the laser beam it creates a peak or pulse of photon emissions over time

These are detected by the PMT and converted to a voltage pulse, known as an event

The total pulse height and area are measured by the flow cytometer and it will correlate directly to intensity of fluorescence for that event

Each event is given a channel number depending on its measured intensity

The more intense the fluorescence, the higher the channel number assigned to the event

47
Q

What’s a gating strategy?

A

To identify cells if interest based n size and granularity

To ignore debris or dead cells

To ignore doublets or triplets or more

Regenerative Medicine (8 decks)

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